Markland 1971

Subtilisins: Primary Structure.
Chemical and Physical Properties

FRANCIS S. MARKLAND. JR . EMIL L . SMITH
I . Introduction . . . . . . . . . . . . . 5 6 2
I1. Historical Background and Development . . . . . .562
I11. Physical. Chemical. and Stability Properties of the Subtilisins . 564
A . Subtilisin Carlsberg . . . . . . . . .564
B . Subtilisin BPN' . . . . . . . . . . 5 8 5
C . Subtilisin Novo . . . . . . . . . .566
D . Subtilisin Amylosacchariticus . . . . . . . 5 6 6
IV . Primary Structure of the Subtilisins . . . . . . . 567
A . General Comparison . . . . . . . . .567
B . Subtilisin B P N . . . . . . . . . .567
C . Subtilisin Novo . . . . . . . . . . 569
D . Subtilisin Carlsberg . . . . . . . . . .569
E . Subtilisin Amylosacchariticus . . . . . . .569
F. Comparison of Sequences . . . . . . . .571
V. Active Site Studies . . . . . . . . . . .575
A . Serine . . . . . . . . . . . . .576
B. Histidine . . . . . . . . . . . . 580
VI . Substrate Specificity and Enzymic Properties . . . . . 5 8 4
A . Protein and Peptide Substrates . . . . . . .584
B . Synthetic Substrates . . . . . . . . .586
C . Transesterification and Transpeptidation . . . . .593
D . Mechanism of Action of the Subtilisins . . . . . 593
VII . Chemical Modification Studies . . . . . . . . .596
A . Lysine Modification . . . . . . . . .596
B. Methionine Modification . . . . . . . .598
C . Tyrosine Modification . . . . . . . . . 599
VIII . Inhibitors: Dye Binding . . . . . . . . . . 602
IX . Other Bacillus Alkaline Proteinases . . . . . . .605
561
562 F. S. MARKLAND, JR., AND E. L. SMITH
X. Practical Uses of Subtilisins . . . . . . . . . go6

A. Protein Sequence . . . . . . . . . . 607
B. Use in Detergents . . . . . . . . . . 608
1. Introduction
Since the last review o i this subjcct appeared in this treatise (1) con-
siderable progress has been made in our understanding of both the struc-
ture and properties of bactcrial proteinases. This chapter will review
only the subtilisins: the diisopropylfluorophosphate (DFP)-sensitive,
extracellular, alkaline proteinaees from Bacillus s u b t i h and other species
of Bacillus. Since most o f the recent work has been on the subtilisins,
major emphasis will be placed on these enzymes with mention of other
alkaline proteinases as applicable. A separate chapter ( 2 ) reviews the
X-ray structure of subtilisin BPN. Other bacterial proteinases are dis-
cussed separately (3).
II. Historical Background and Development
The extracellular alkalinc proteinase, subtilisin, was discovered by

Linderstrgm-L.ang and Ottesen ( 4 ) during an investigation of the con-
version of ovalbumin to plakalbumin. Subsequent work in the Carlsberg
laboratory led to the isolation and crystallization of this enzyme, sub-
tilisin Carlsberg (5-7). Later, another type of subtilisin, BPN’, was
isolated and crystallized (8). Since the earlier review by Hagihara (1)
described the preparation of both subtilisins Carlsberg and BPN’ only
the isolation and characterization of other subtilisins are included here.
1. B. Hagihara, “The Enzymes,” 2nd ed.. Vol. 4, p. 193, 1960.

2. J. Kraut, “The Enzymes.” 3rd ed.. Vol. 111, Chapter 15, 1971.
3. H. Matsubara and J. Feder, “The Enzymes,” 3rd ed.. Vol. 111, Chapter 20,
1971.
4. K. Linderstrom-Lang and M. Ottesen. Nature 159, 807 (1947).
5. A. V. Giintelberg and M. Ottrsen, Nature 170, 802 (1952).
6. A. V. Giintelberg, Compt. Rend. Trav. Lab. Carlsberg, Ser. Chim. 29, 27
(1954).
7. A. V. Giintelberg and M. Ottesen, CompL. Rend. Trav. Lab. Carlsberg, Ser.
Chim. 29, 36 (1954).
8. B. Hagihara, H. Matsubara, M. Nakai, and K. Okunuki, J . Biochm. (I’okvo)
45, 185 (1958).
16. SUBTILISINS 663
In 1960, Ottesen and Spector (9) reported on the characteristics of yet

another extracellular alkaline bacterial proteinase, designated subtilisin
Novo. Although it converted ovalbumin to plakalbumin like subtilisin
Carlsberg, it exhibited different chromatographic and kinetic properties.
Subtilisin Amylosacchariticus was purified by Tsuru et al. (10) by a
combination of ammonium sulfate precipitation ; chromatography on Duo-
lite A-2, CM-cellulose, and DEAE-Sephadex; and crystallization from
tris-HC1-calcium acetate buffer a t pH 8.5. Little difference was noted in
the enzymic properties of this alkaline proteinase and other subtilisins.
This proteinase as well as subtilisins B P N (11, It?), Carlsberg (Y), and
Novo (9),all have esterase activity and are inhibited by DFP.
Keay and Moser (13) have prepared additional alkaline proteinases
from B . subtilis (NRRL B3411), B . licheniformis, and B. pumilis by
acetone precipitation, batch treatment with DEAE-cellulose, chroma-
tography on Duolite C-10 ion exchange resin, reprecipitation, dialysis,
and lyophilization. They showed that these alkaline proteinases can be
classified into two groups based on their amino acid compositions, sero-
logical cross-reactions, and ratio of esterase to proteinase activity. Group
A includes subtilisin Carlsberg and the enzymes from B. Zicheniformis
and B . pumilis,whereas group B includes subtilisins NOVO, BPN', Amylo-
sacchariticus and NRRL B3411. They suggested that the name Bacillo-
peptidase be given t o these enzymes. Inasmuch as the name subtilisin is
well established, we shall follow the binomial system already in wide use
by maintaining the generic term subtilisin followed by the name of the
specific variety of enzyme under study (14).
Welker and Campbell (15) have indicated that the organism from
which subtilisin BPN' is derived should be called Bacillus amylolique-
faciens and is a different species from B . subtilis. These authors con-
cluded that the relatively large differences reported by Smith et a,?. (16)
in the sequences of subtilisins B P N and Carlsberg were due to the fact
that they were derived from different species of Bacillus and not different
strains of the same species as originally believed.
9. M. Ottesen and A. Spector, Compt. Rend. Trav. Lab. Carlsberg 32, 63 (1960).
10. D. Tsuru, H. Kira, T. Yamamoto, and J. Fukumoto, Agr. Biol. Chem. (Tokyo)
30, 1261 (1966).
11. H. Matsubara, B. Hagihara, M. Nakai, T. Komaki, T. Yonetani, and K .
Okunuki, J . Biochem. (Tokyo) 45, 251 (1968).
12. H. Matsubara and S. Nishimura, J . Biochem. (Tokyo) 45,603 (1968).
13. L. Keay and P. W. Moser, BBRC 34, 600 (1969); L. Keay, P. W. Moser, and
B. S. Wildi, Biotechnol. Bioeng. 12, 213 (1970).
14. S. A. Olaitan, R. J. DeLange, and E. L. Smith, JBC 243,6296 (1988).
15. N. E. Welker and L. L. Campbell, J . Bacleriol. 94, 1124 and 1131 (1967).
16. E. L. Smith, F. S. Markland, C. B. Kasper, R. J. DeLange, M . Landon, and
W.H. Evans, JBC 241, 5974 (1986).
111. Physical, Chemical, and Stability Properties of the Subtilisins
A. SUBTILISINCARLSBERG
Guntelberg and Ottesen (7) reported that subtilisin Carlsberg had an

isoelectric point of 9.4 and a typical protein ultraviolet absorption spec-
trum. During electrophoresis experiments it was found that the protein
was more stable a t acid pH (pH 5.3 or 6.5) than a t alkaline p H (pH
8.1 and 9.5) where autolysis of the enzyme occurs. I n the alkaline range
the enzyme appears t o be more stable than trypsin or chymotrypsin.
Furthermore, the proteinase is stable for several months in the lyo-
philized state, in dialyzed solution kept frozen at -lO"C, or in glycerol
solution a t room temperature; the activity is rapidly lost a t pH values
below 5. The sedimentation coefficient was reported to be s20,w= 2.85 S
( 7 ) , and the amino-terminal sequence of the diisopropylphosphoryl
(DIP) enzyme as NH2-Ala-Glx (1'7). Table I presents some of the
TABLE I
OF PHYSICOCHEMICAL
COMPARISON PROPERTIES
OF THE SUFITILISINS
Amylosac-
BPN' Carlsberg Novo chariticus
Propertmy (18-20) (7, 17, 21,221 (9,141 (10,23, 24)
E2nm 11.7 8.6 11.7 11.9

Partial specific volumea 0.731 0.725 0.731 0.722
Molecular weight
Sedimentation 26,000 27,40@ - 28,000
equilibrium
Amino acid sequence 27 ,537 27 ,277 27,537 27,671
s2o.w
2.77 2.85 - 2.71
PI 7.8 9.4 9.1 7.8
f/fQ 1.18 - - 1.04
NHp-terminalresidues Ala Ala Ala Ala
Elemental analysis (%P N 17.3 17.0.5 17.3 17.01
Elemental analysis S 0.58 0.59 0.58 0.46
Calculated from the amino acid composition.
* Phosphorous content of DIP enzyme.
Calculated from the amino acid sequence.
17. M. Ottesen and C. G. Schellman, Compt. Rend. Trav. Lab. Carlsberg, Ser.
Chim. 30, 157 (1957).
18. H. Matsubara, C. B. Kaaper, D. M. Brown, and E. L. Smith, JBC 240, 1125
( 1965).
16. SUBTILISINS 565
TABLE I1
AMINOACID COMPOSITIONS OF SUBTILISINS
Carlsberg. Amylosacchariticusc
Amino acid BPN’(I (19) (a1) Novob (14) (94.6)
Lysine 11 9 11 8
Histidine 6 5 6 6
Arginine 2 4 2 4
Tryptophan 3 1 3 3
Aspartic acid 11 9 11
26
Asparagine 17 19 17
T hreonine 13 19 13 16
Serine 37 32 37 40
Glutaniic acid 4 3 4
15
Glutamine 11 7 11
Proline 14 9 14 13
Glycine 33 35 33 33
Alanine 37 41 37 30
Valine 30 31 30 25
Methionine 5 5 5 4
Isoleucine 13 10 13 16
Leucine 15 16 16 15
Tyrosine 10 13 10 12
Phenylalanine 3 4 3 3
Cysteine, cystine 0 0 0 0
Total 275 274 275 275
From the amino acid sequence.

* From analysis of tryptic peptides; the compositions are t,he same as the tryptic p e g
t,ides from subtilisin BPN’.
From analysis of tryptic and cyanogen bromide peptides.
physicochemical properties of subtilisin Carlaberg. The amino acid

composition is given in Table I1 (21, 2 2 ) .
B. SUBTILISIN
BPN’
Subtilisin BPN’ was shown to have essentially the same stability

characteristics as subtiliein Carlsberg and also to be stabilized by Ca2+
19. F. S. Mark1:ind and E. L. Smith, JBC 242, 5198 (1967).

20. C. U. Kasper, H. Matsubara, and E. L. Smith. JBC 240, 1131 (1965).
21. R. J . DeLange and E. L. Smith, JBC 243,2134 (1968).
22, G. Johansen and M. Ottesen, Compt. Rend. Trav. Lab. Carlsberg 34, 199
( 1964).
23. D. Tsuru, H. Kira. T. Yamamoto, and J. Fukumoto, Agr. BWZ. Chem.
(Tokyo) 31, 330 (1967).
24. F. S. Markland, M. Kurihara, and E. L. Smith, unpublished studies (1970).
566 F. S. MARKLAND, J R . , AND E. L. SMITH
and other salts (11). Thc proteinase was shown to be a single polypep-
tide chain of molecular weight 27,600 having an isoelectric point of 7.80
(18).The amino acid composition is given in Table I1 (19, 22) and the
amino- and carboxyl-terminal sequences of the D I P enzyme were identi-
fied as NH,--Ala-Glx and Ala-Gln-COOH, respectively ( 2 0 ) . These re-
sults agree with carlier reports by the Japanese workers who showed that
the enzyme had a molecular weight of about 30,500 with alanine as the
amino-terminal residue (25).
C. SUBTILISINNovo
Subtilisin Novo was shown to be similar in physicochemical proper-

ties to subtilisin Carlsberg (91. The isoelectric point of Novo was, how-
ever, somewhat lower than that of subtilisin Carlsberg. The stability
characteristics of subtilisin Novo are similar to the other subtilisins and
all are extremely acid labile. However, the remarkable resistance of
these proteinases to a variety of denaturing agents such as 6 M urea and
50% ethanol has been noted (261, and these enzymes are stable as well
to a variety of detergents such as sodium dodecyl sulfate, and sodium
tripolyphosphate (27).
D. SUBTILEISAMYLOSACCHARITICUS
Tsuru and his co-workers (10 reported that subtilisin Amylosac-

chariticus was much less soluble in neutral solution than the other sub-
tilisins, and could be crystallized during dialysis against weak salt solu-
tions between pH 6.5 and 8.5 if the enzyme concentration was higher
than 0.5%. Otherwise the stability properties of this enzyme were similar
to those already reported for the other subtilisins. The sedimentation
coefficient was determined to be 2.89 S and from approach to sedimcnta-
tion equilibrium, the molecular weight was reported to be 22,700. End
group analyses showed alanine to be the amino-terminal residuc ( 2 3 ) .
Reinvestigation (24) of the molecular weight by approach to sedimen-
tation equilibrium gave a value of 28,000 for the DIP derivative indicat-
ing that autolysis may have resulted in the low molecular weight ob-
tained by the Japanese workers ( 2 3 ) .The isoelectric point is at p H 7.8
25. H. Matsubara and S.Nishimura, J . Biochem. (Tokyo)45, 413 (1958).
26. A. Gounaris and M. Ottesen, Compl. Rend. Truv. Lab. Carlsberg 35, 37
(1965).
27. D. Tsuru, Kagaku To Kogyo (Osaka) 43, 199 (1969); cited in CA 71, 79183j
(1969).
16. SUBTILISINS 567
(identical to that of subtilisin BPN’), and the amino acid composition

(24) is more like that of BPN’ than of Carlsberg in agreement with the
classification of Keay and Moser (13).
Tables I and I1 present the physicochemical properties and amino
acid compositions, respectively, of the various subtilisins.
IV. Primary Structure of the Subtilirinr
A. GENERAL
COMPARISON
Although many of the physical properties of the subtilisins are similar

(see Table I) their amino acid compositions show considerable differ-
ences [see Table I1 (14, 19, 21, 22, 24)]. Peptide mapping indicated
definite similarity between subtilisin BPN’ and Novo but also showed
that they were different from subtilisin Carlsberg ($8).This was sub-
stantiated by the amino acid analyses.
A feature of the amino acid compositions of the subtilisins is the com-
plete absence of cysteine or cystine in contrast to the high content of
disulfide bridges in the several pancreatic proteinases ( $ 9 4 4 ) .
B. SUBTILISIN
BPN’
The sequence of subtilisin BPN’ was deduced from studies of the tryp-
tic ($53,chymotryptic (36),peptic (37), and cyanogen bromide digests
(19). The complete sequence is shown in Fig. 1.
The protein consists of a single polypeptide chain of 275 residues de-
void of any disulfide bridges, There is no apparent homology with the
sequences of the pancreatic proteinases (16). The serine residue reactive
28. J. A. Hunt and M. Ottesen, BBA 48,411 (1961).

29. 0. Mike:, V. HoleyZjovskf, J. TomlAek, and F. &om, BBRC 24, 346 (1966).
30. K. A. Walsh and H. Neurath, Proc. Natl. Acad. Sci. U. S. 52, 884 (1964).
31. J. R. Brown and B. S. Hartley, BJ 101,214 (1968).
32. B. S. Hartley, J. R. Brown, D. L. Kauffman, and L. B. Smillie, Nature 207,
1157 (1965).
33. B. Meloun, V. Kostka, J. VanBEek, 1. H u h , and F;Sorm, Collection Czech.
Chem. Commun. 31, 321 (1966).
34. D. M. Shotton and B. S. Hartley, Nature 225,802 (1970).
35. C. B. Kasper and E. L. Smith, JBC 241,3754 and 3771 (1966).
36. F. S. Markland, G. Kreil, B. Ribadeau-Dumas, and E. L. Smith, JBC 241,
4642 (1966).
37. F. S. Markland, B. Ribadeau-Dumas, and E. L. Smith, JBC 242, 5174 (1967).
Th r Ile Pro Leu Asp Lvs Val Gln Ala PheLys Ala
-
NH -Ala-Gln-Ser-Val-Pro-Tyr-Gly-Val-Ser-Gln~lle-Lys-Ala-Pro-Ala-Leu~His-Ser-Gln-Gly-Tyr-Thr-Gly-Ser-Asn-Val~Lys-Val-Ala-Val~
2 10 a 30
Leu Thr Gln Ala Am Val Phe Ala Gly Ala Tyr Asn Thr
Ile-Asp-Ser-Gly-l le-Asp-Ser-Ser-His-Pro-Asp-Leu-Lys-Val-Ala-Gly-Gly-Ala-Ser-Met-Val-Pro-Ser-Glu-Thr-Pro-Asn~Phe~Gln-Asp-
40 m m
Gly Gly ASP lhr Thr Val Ser
Asp-Asn-Ser-His-Gly-Thr-His-Val-Ala-Gly-Thr-Val-Ala-Ala-Leu-Asn-Asn~Ser-l le-Gly-Val-Leu-Gly-Val-Ala-Pro-Ser-Ser-A~-~u-
70 m 9Ll
A m Ser Ser Ser Gly Val Ser Thrlhr Gly
Ty~-Ala-Val-Lys-VaI-Leu-Gly-Asp-Ala-Gly-Ser-Gly-Gln-Tyr-Ser-Trp-Ile-I le-Asn-Gly-l le-GI uTrp-Ala-lle-Ala-Asn-Asn-hret-Arp-
Im 110 120
Ala Thr Me1 Gln Am Tyr Arg
Val-I le-Asn-Met-Ser-Leu-Gly-Gly-Pro-Ser-Gly-Ser-Ala-Ala-Leu-Lys-Ala-Ala~Val-Asp-Lys-Ala-Val-Ala-Ser-Gly-Val-Val-Val-Val~
130 la im
Ser Asn Ser Thr Asn Ile Ala Asp
Ala-Ala-Ala-Gly-Asn-Glu-Gly-Ser-Thr-Gly-Ser-Ser-Ser-Thr-Val-Gly-lyr-Pro-6ly-Lys-Tyr~Pr~Ser-Val-l le-Ala-Val-Gly-Ala-Val-
1M 170 180
Asn Ser Asn Ala Glu Ala Gly Val Tyr 1~r
Asp-Ser-Ser-Asn-Gln-Arg-Ala-Ser-Phe-Ser-Ser-Val-Gly-Pro-Glu-Leu-Asp-VaI-Met-Ala-Pro-Gly-Val-Ser-l le-GIn-Ser-Thr-Leu-Pro-
190 200 210
Thr Thr Ala Thr Leu
Gly-Asn-Lys-Tyr-Gly-Ala-Tyr-Asn-Gly-Thr-Ser-Met-Ala-Ser-Pm-His-Val-Ala-Gly-AIr-Ala-Ala-Leu-I le-Leu-Ser- Lys-His-Pro-Asn-
220 rm 24l
l e u Ser Ala Ser Asn Arg Ser Ser Ala Tyr Ser
Trp-Thr-Asn-lhr-Gln-Val-Arg-Ser-Ser-Leu-Gln-Asn~hr~Thr-Thr-Lys-Leu-Gly-As~-Ser-Phe-Tyr-Tyr-Gly-Lys-Gly-Leu- Ile-Asn-Val-
2% 2M 210
Glu
Cln-Ala-Ala- Ala-GlnCOOH
71%
FIG.1. Comparison of amino acid sequence of subtilisins BPN' and Carlsberg
(42).The continuous sequence is that of subtilisin BPN. The residues which differ
in subtilisin Carlsberg are given above the corresponding residue. The dash at
residue 56 indicates that the corresponding residue is lacking in the Carlsberg en-
zyme. The active Ser 221 is denoted by asterisk.
with DFP is a t position 221 and the sequence around that serine (Thr-
Ser+-MetrAla), as originally pointed out by Sanger and Shaw (38),is
different from that around the reactive serine in the mammalian pan-
creatic proteinases (see Section V,A) .
There are several sequences in the polypeptide chain which appear to
be repeated. These may have some relationship to the evolutionary his-
tory of the subtilisins and will be discussed in Section IV,F.
Although the possible significance is presently unknown there are
many di-, tri-, and, in one case, tetrapeptide (residue 147 through 150,
Fig. 1) repetitions of the same residue. The residues appearing in these
repeating sequences are Ala (six Ala-Ala sequences, three of which are
Ala-Ala-Ala) , Val (one Val-Val-Val-Val sequence), Ser (six Ser-Ser
sequences, including one Ser-Ser-Ser) , Thr (one Thr-Thr-Thr se-
quence), Gly (two Gly-Gly sequences), Ile (one I l e I l e sequence), and
38. F.Sanger and D. C. Shaw, Nature 187,872 (1960).

16. SUBTILISINS 569
Tyr (one Tyr-Tyr sequence). Additionally, there are many Ser-Thr or

Thr-Ser sequences.
C. SUBTILISINNovo
The chromatographic behavior and compositions of the 14 expected

tryptic peptides from subtilisin Novo have been reported (14). Since
these were identical in all respects to those from subtilisin BPN', it was
concluded that the two enzymes are identical. Furthermore, comparison
of various enzymic and chemical properties all indicated the identity of
the Novo and BPN' enzymes (22,28,39-41) ,
D. SUBTILISINCARLSBERG
The complete sequence of subtilisin Carlsberg (@) was determined

from the tryptic (21, 43) and chymotryptic peptides (44, 46) (see Fig.
1). The protein contains 274 residues in a single polypeptide chain and
differs from subtilisin BPN' in 85 positions including one deletion. Al-
though the total base composition of subtilisin BPN' (11 lysine and 2
arginine residues) and Carlsberg (9 lysine and 4 arginine residues) are
identical, the base substitutions are not in the same positions in the se-
quence ; these differences in base positions contribute to the radically
different tryptic peptides of the two enzymes (4.2).
E. SUBTILISIN
AMYLOSACCHARITICUS
The partial sequence of subtilisin Amylosacchariticus (24) is shown

in Fig. 2 in comparison with the complete sequence of subtilisin B P N .
Tryptic hydrolysis and cyanogen bromide cleavage have yielded frag-
ments which account for the approximately 275 residues in the protein.
The part of the sequence already determined shows great similarity to
that of subtilisin BPN', although there are some features which resemble
subtilisin Carlsberg.
39. A . N. Glazer, JBC 242, 433 (1967).
40. ,4.0. Barel and A . N. Glazer, JBC 243, 1344 (1968).
41. J. Drenth and W. G. J. Hol, J M B 28,543 (1967).
42. E. L. Smith, R. J. DeLange, W. H. Evans, M. Landon, and F. S. Markland,
JBC 243, 2184 (1968).
43. R. J. DeLange and E. L. Smith, JBC 243,2143 (1988).
44. M. Landon, W. H. Evans, and E. L. Smith, JBC 243,2165 (1868).
45. W. H. Evans, M. Landon, and E. L. Smith, JBC 243,2172 (1968).
NHI- .~(Gln~Ser,Val.Pro)Tyr-Gly(Ile~Ser~Gln~Ile)Lys(Ala~Pro,Ala~~u~His)(Ser,Gln,Gly,Tyr)(Thr.Gly~Ser,ilsn.Val)Lys~al.Ala~~’al.
NH~-Ala-Gln-Ser-Val-Pro-Tyr-Gly-Val-Ser-Gln-Ile-Lys-.4la-Pro-Ala-~u-H~-Ser-Gln-Gly-Tyr-Thr-Gly-Ser-Asn-Val-I,ys-Yal-~~la-~‘al- Ql
10 20 30 -3
0
Ile.Asp.Ser,Gly.Ile.Asp,Ser. Ser.His. Pro.Asp,Leu)- (Yal..\la,Gly. Gly,Ala.Ser.Ser.Val.Pro.Ser.Glu.Thr.Pro.Tyr,Phe.GIn.As~
Ile-AspSer-Gly-Ile-.4spSer-Ser-H~-Pro-,bpLeu- Lys-Val-hla-Gly-Gly-AlaiSer-Met-Val-Pro-Ser-Glu-Thr-Pro-Aen-Phe-GIn-Asp-
40 50 60
Gly. Asn.Ser.His*.Gly. Thr.His, Ile. hla. Gly.Thr.Val,Ala. Ma. Leu, .~n.Asn.Ser.Ile.Glg.Val.Leu,Gly.Ser,hla,Pro.Ser,Ser,Ala,Le~~,

AspAsn-Ser.His*-Gly-Thr-His-Val-.~~-Gly-Thr-Yal-Ala-.4~-I~u-.~n-.4sn-Ser-Ile-Gly-Val-l.eu-C.ly-Val-.4la-Pro-Ser-Ser-.4la-Lei1-
70 80 90
Tyr)(Ala.Val) Lys ~Yal.I~u.Thr,Asp,Ile.Gly.Ser.Gly.Gln.Tyr.Ser.Trp.Ile)(Ile,~n.Gly.Ile,Glu)Trp(.\la,Ile,Ser..\sn,Asn)~let

(.hp.
Tyr-Ala-Yal-Lys-Val-Leu-Gly-.bp-Ala-Gly-Ser-Gly-Gln-Tyr-Ser-Trp-Ile-Ile-Asn-Gly-Ile-Glu-Trp-Ala-Ile-Ala-.hn-Asn-~et-.~say
100 110 120
Val, Ile,A m ) Met (Ser,Leu,Gly. Cly.Pro.Ser.Gly,Ser.Thr..Ua.Leu)Lgs-Thr-Val-Val-ilsspLys(rUa. Val,Ala.Ser.Gly.Val,Val.~al,
Ile.
Val-Ile-Asn-Met-Ser-Leu-Gly-G
ly-Pro-Ser-Gly-Ser-Ala-~~l~u-I,ys-Ala-Ala-~’al-ils~-I,ya-A~-Yal-Ala-Ser-Gly-Yal-Yal-Yal-Val-
130 140 150
Ala,Ala,Ala.Gly.Asn,Glu,Gly.Ser.Ser.Gly.Ser.Ser.Ser.Thr~Val.Gly,Tyr~Pro.~)Lys(Tyr.Pro,Ser~Val~Ile..~la~Thr~Gly~Ala.Yal.
Ala-Ala-Ala-Gly-.~n-Glu-Gly-Ser-Thr-Gly-Ser-Ser-Ser-Thr-Val-Gly-Tyr-Pro-Gly-Lys-Tyr-Pro-Ser-~al-Ile-.4la-Val-GIy-.4la-Yal-
160 170 180 r
Asp,Ser.Ser,.4sn,Gln)Arg(Ala,Ser,Phe.Ser.Ser.Ser,Gly,Ala.Glu.Leu.ilsp,Yal)Jlet
(.Ua.Pro.Gly )(Yal,Ser)Ile-Gln-Ser-Thr-1,eu-Pro- P
~aySer-Ser-~n-Gln-~g-Ala-Ser-Phe-Ser-Ser-V~-Gly-R~Gl11-I~eu-.4sp-Val-.~et-Ala-Pro-Gly-~al-~~r-Ile-~~In-Ser-Thr-I.eu-Pro-
190 200 210
~Val.Ala.Gly,Ala.Ala,Ala.Leu)(Ile,Leu,Ser)Lys(His.Pro.Asn.
Gly-Gly-Thr-Tyr-Gly-.4Ls-Tyr(Asn,Gly.Thr,Ser*)~~et(Ala.Thr.Pro.His)
Gly- Asn-Lys-Tyr-Gly-Ala-Tyr-Asn-Gly-Thr-Ser*-~let-Als-Ser-Pro-His-Val-Ala-Gly-Ala-A1a-.4la-l~eu-Ile-I~u-Ser-I,y~-His-Pro-Asn-
220 230 240
Trp)(Thr.hsn ) (Ala.Gln,Val)Arg(Asn,Arg)
(Leu,Gln,Ser,Thr,Ala,Thr.?lr)
(Leu.GIy.hsp)(Ser.Phe.Tyr.Tyr.C.ly)I.ys(Gly.I.eo. Ile..bn.Val
Trp-Thr- ~bn-Thr-Gln-Val-Ara-Ser-Ser-Leu-Gln-Asn-Thr-Thr-Thr-Ly~l~ii-(;ly-.~p-Ser-Phe-Tyr-Tyr-C.Iy-l.ys-(;ly-Leu-Ile-.49n-Ya1-
250 260 270
Gln)Ala-Ala-Ah-GlnCOOH
Gln-Ala-Ala-Ala-GlnCOOH
275 m
FIG.2. Comparison of amino acid sequence of subtilisins RPN’ and Amylosacchariticus (944).The lower se- r
quence is that of BPN. Residues in subtilisin Amylosacchariticus whose sequences have not been deter-
mined are enclosed in parentheses. However, due to the extensive homology the arrangement of many oi
the residues within the parentheses is probably correct. The active His 64 and Ser ‘221 are denoted by aster- x
isks. Apparent differences are indicated in boldface. The two enzymes apparently differ by at least 28 residues.
16. SUBTILISINS 571
There are only 4 methionine and 12 basic residues in subtilisin Amylo-

sacchariticus as compared to 5 and 13, respectively, in the other sub-
tilisins. The 4 methionine residues are in the same positions as the 4
constant methionine residues in subtilisins BPN’ and Carlsberg (resi-
dues 119, 124, 199, and 222; Fig. 1 ) . The missing base appears to be at
residue 213 where lysine is replaced by threonine in subtilisin Carlsberg.
The other bases are in the same positions as in subtilisin BPN’ except
for residue 249 where there appears to be an arginine for serine replace-
ment as in subtilisin Carlsberg and residue 256 where lysine is replaced
by an unknown residue.
One interesting substitution occurs in the region around the reactive
Ser 221 where the sequence is the same in subtilisin BPN’ and Carlsberg.
This change in subtilisin Amylosacchariticus involves the substitution of
threonine for serine a t residue 224.
F. COMPARISON
OF SEQUENCES
In comparing the sequences of subtilisins B P N and Carlsberg and the

partial sequence of subtilisin Amylosacchariticus, certain generalizations
can be made. Although the subtilisins resemble the pancreatic proteinases
in many properties, e.g., inhibition by D F P (la,l 7 ) , involvement of a
histidine residue (4S),and pH optima, there appears to be no similarity
in their amino acid sequences. The absence of disulfide bonds and the
high content of glycine, alanine, serine, and valine in the subtilisins is
particularly noteworthy. Furthermore, the sequences around the reactive
histidine and serine residues, which will be discussed in a later section,
do not resemble those in the vertebrate enzymes; and the positioning and
sequence around the aspartic acid residue involved in the active site of
the subtilisins (4‘7) are completely different from these in the mammalian
proteinases (48) (Table 111).Thus, the animal and bacterial proteinases
would seem to have evolved independently, although a similar mecha-
nism of action seems to have been the end result [also, see Smith (@)I.
Of the 275 residues in the polypeptide chains there are 84 differences
between subtilisins BPN’ and Carlsberg plus a deletion of one residue in
subtilisin Carlsberg (as indicated by the dash a t residue 56 in Fig. 1 ) .
This position has been chosen in order to minimize the number of muta-
46. F. S. Markland, E. Shaw, and E. L. Smith, Proc. Natl. Acad. Sci. U. S . 61,
1440 (1968).
47. C. S. Wright, R. A. Alden, and J. Kraut, Nature 221,235 (1969).
48. D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature 221, 337 (1969).
49. E. L. Smith, “The Enzymes,” 3rd ed., Vol. I, Chapter 6,1970.
TABLE I11
SEQUENCES
AROUND THE ASPARTYLRESIDUESIN ACTIVE SITESO
Enzyme Sequence'
Chymotrypsin A (bovine) hr-Leu-Leu-Lys

Ser-Leu-Thr-Ile-Asn-Asn-Asp*-Ile-T
96 100 102 105 107
Chymotrypsin B (bovine) Ile-Leu-Thr-Val-Arg-Asn-Asp'-Ile-T hr-Leu-Leu-Lys
96 100 102 105 107
Trypsin (bovine) Ser-Asn-Thr-Leu-Asn-Asn-Asp*-Ile-Met-Leu-Ile-Lys
96 100 102 105 107
Thrombin (bovine) -Glx-Asx-Leu-Asp-Arg-Asp*-Ile- Ala-Leu-Leu-Lys
97 100 102 105 107
Elastase (porcine) AspVal- Ala-Ala-Gly-T yr-Asp*-Ile- Ala-Leu-Leu- Arg
98 9999A99B 100 102 105 107
Subtilisin BPN' Val-Ala-Vsl-Ile-Asp'-Ser-Gl y-Ile-Asp
28 30 32 35 36
Subtilisin Carlsberg Val-Ala-Val-Leu-Asp'-T hr-Gly-Ile-Gln
28 30 32 35 36
Subtilisin Amylosacchariticus (Val,Ala,Val,Ile,Asp',Ser,Gly,Ile, Asp)
28 30 32 35 36
4 Residue numbers are below the sequence using the numbering of chymotrypsino-
gen A for the mammalian proteinases. The active aspartic acid residue is marked by
an asterisk.
* For identificationin mammalian enzymes,see Hartley el al. (34,48);in the subtilisins,
see Smith et u2. (4S),Wright et al. (47), and Fig. 2.
tional events in the nucleotide codons in this region. Nevertheless, the

addition or deletion must have occurred between residues 50 and 59 in
view of the excellent homology of the sequences of the two subtilisins on
eibher side of this portion of the sequence.
Table IV gives the amino acid composition of subtilisin BPN' and in-
dicates the replacements in the Carlsberg enzyme. From the presently
assigned triplet nucleotide codons for the amino acids ( 5 0 ) , 61 of the
changes can be ascribed to single base changes and 23 to double base
changes.
I n view of the close similarity of the enzymic properties of the two
subtilisins (39, $0) it was somewhat surprising to find that they differed
in 30% of the structure. The many similarities in their properties suggest
that the polypeptide chain conformations are very similar, if not identi-
cal, for the two subtilisins and that most substitutions would be con-
50. F. H. C. Crick, Cold Spring Harbor synp. Quant. Bwl. 31, 1 (1966).
16. SUBTILISINS 573
TABLE IV
AMINOACID SUBSTITUTIONS
IN SUBTIUSINS
Subtilisin BPN'
Changes Subtilisin Carlsberg changed

Residue Total (%I residue (No.)"
Lysine 11 36 Asn (2),Thr (l),TYR (1)

Histidine 6 17 Gln (1)
Arginine 2 0
Aspartic acid 11 45 GLN (l),Gly (l),Glu (l),Ser (2)
Asparagine 17 41 Tyr (l),Asp (I), Ser (31,GLY (11, ALA (1)
Glutamic acid 4 25 SER (1)
Glutamine 11 64 Leu (1); THR (11, SER (21,ASN (I),
TYR (l),Glu (1)
Threonine 13 46 LYS(I), Ala (2), Ser (3)
Serine 37 49 Thr (41,Pro (l), Ala (31, Gly (31, VAL (11,
Arg (21,Asn (4)
Proline 14 43* ASP (2),Ala (31, deletion (1)
Glycine 33 12 ASN (I), Ala (2),THR (1)
Alanine 37 22 LYS (I),Val (11, Ser (21,Thr (3), GLN (1)
Valine 30 13 Ile (2),TYR (11, Ale (1)
Isoleucine 13 38 Leu (I), Thr (2), Val (2)
Leucine 15 20 Val (I),Met (I), TYR (1)
Methionine 5 20 PHE (1)
Tyrosine 10 20 Phe (I), LEU (1)
Phenylalanine 3 33 ASN (1)
Tryptophan 3 67 Gly (11, Leu (1)
Total 275 30.9
Residues in capital letters involve a t least two base changes in the triplet codon;
other changes can be accounted for by single base changes.
Includes deletion.
servative in nature. It is evident from the information in Table I V that

a t least for the hydrophobic residues most of the substitutions are con-
servative involving residues of the same type. Furthermore, the complete
three-dimensional structure of subtilisin BPN' shows that all substitu-
tions with but one exception occur in exterior chain segments (4'7) ; the
exception is Ile 31 which is Leu in subtilisin Carlsberg. The side chains
of 75 of the 84 substituted residues are pointed outward, and of the 9
inward-pointing side chain substitutions, 8 are conservative. The radical
change is Ser 88 which is Val in Carlsberg. A more detailed description
of the X-ray work on subtilisin B P N appears in this volume (9).
Most of the amino acid substitutions are present throughout the linear
sequence ; however, there are several fairly long segments of' the sequence
574 F. S. MARKLAND, JB., AND E. L. SMITH
which show no substitution. One of these occurs around the reactive Ser
221 extending from residues 218 through 240, and another is around the
reactive His 64 extending from residues 64 through 75. As noted above,
there is a conservative substitution of Ser 224 by Thr in subtilisin
Amylosscchariticus.
Comparison of the sequences of subtilisins B P N and Carlsberg shows
the presence of similar sequences in different portions of the molecules.
These are obvious in either subtilisin individually but are even more ap-
parent when both protein sequences are considered together (Fig. 3 ) . In
addition to those shown in the figure, there are a number of tripeptide
(A)
67 75
Hie-Val-Ala-Gly-T hr-Val-Ala-Ala-Leu
His-Val-Ala-Gly - Ala-Ala-Ala-Leu
226 229 230 233
(B)
82 Val-Ser
Leu-Gly-Val-Ala-Pro-Ser-Ser-Ala-Leu-Tyr-Ala- Val-Lya
Leu-Gly - Gly-Pro-Ser-Clly-Ser-Ala - Ala-Leu-Lya
126 127 128Ala Thr 134 Met 136
(C)
88
77 Thr Thr Val
Aan-Ser-Ile-Gly-Val-Leu-Gl y-Val- Ala-Pro-Ser-Ser
Ser-Thr- Val-Gly-Tyr-Pro-Gly-Lys-T yr-Pro-Ser-Val
Aan Ile Ala Asp 174
163
(D) (E)
45
39 42 Val 49
HbPro-Asp-Leu Ala-Gl y-Gly-Ala-Ser
HbPro-Am-Trp Leu-Gly-Gly-Pro-Ser
238 Leu 126 Ala 130
241
(F) (GI
73 A8p 77 167 Ah 171
Ala-Ala-Leu-Aan-Aen Tyr-Pro-Gly - Lye-Tyr
Ala-Ala-Val-AspLys Leu-Pr o-Gly- Am-Ly a-T yr
Gln A8n Tyr Thr Thr 214
137 141 209
FIG.3. Segments of the sequences of subtilisins B P N and Carlsberg which ap-
pear to involve repetition of similar sequences (4.9). Residues shown in boldface are
identical; residues shown in italics would appear to represent conservative replace-
ments. The continuous sequences are BPN' or both subtilisins; residues above or
below these sequences represent differences in subtilisin Carlsberg.
16. SUBTILISINS 575
repetitions; e.g., Ala-Ala-Ala (residues 151-153, 230 through 232, and

272 through 274), Val-Lys-Val (residues 26 through 28 and 93 through
95), Gly-AsnSer (residues 154 through 156 and 157 through 159 in
Carlsberg) and Val-Leu-Gly (residues 81 through 83 and 95 through
97 in BPN’) .
The presence of repetitive sequences in other proteins has been inter-
preted as involving an extension of shorter peptide chains by a process
of gene duplication followed by later mutational events (49). Such in-
stances appear well documented for the ferrodoxins (61, 62) and for the
7-globulins (53). Thus it appears that the long polypeptide chain of 275
residues in the subtilisins may have evolved by a duplicated lengthening
of the DNA genome.
There are additional features of the sequences in Fig. 3 that merit
comment. First, the repetitions are not in exact linear order but for some
segments appear to be in inverse order, For example, if we consider the
sequences in D, A, and B (Fig. 3 ) , the duplicated sequences are in the
order: residues 39 through 42, 67 through 75, and 82 through 94 in the
NH,-terminal part of the molecule, but they are in the inverse order:
residues 238 through 241, 226 through 233, and 126 through 136 in the
COOH-terminal part. Second, some parts of the sequence are repeated
two or more times; e.g., residues 82 through 88 are repeated not only by
residues 126 through 131 but also by residues 168 through 174 (Fig. 3,
B and C) . The finding that the numbers of residues between the repeated
segments vary and that the repetitions of some segments occur in inverse
order indicates that the subtilisins may have evolved by a complex
mechanism with duplications of the sequence occurring several times.
Hopefully, more insight into the origin and evolutionary development
of these enzymes will be obtained as the sequences of subtilisin Amylo-
sacchariticus and other homologous subtilisins become known.
V. Active Site Studies
A. SERINE
The involvement of serine in the active site of subtilisins Carlsberg

(7) and BPN’ (12) was first indicated by the finding that they were in-
51. H. Matsubara. R. M. Sasaki. and R. K. Chain, Proc. Natl. Acad. Sci. U. s.

57, 439 (1987).
52. R.V. Eck and M . 0. Dayhoff, Science 152,363 (1966).
53. R. L. Hill, R. Delaney. R. E. Fellows, Jr., and H. E. Lebovitz, Proc. Natl.
Acad. Sci. U.S. 56, 1762 (1966).
576 F. S. MARKLAND, JR., A N D E. L. SMITH
activated by DFP. Tsuru and co-workers also showed that DFP is a

potent inhibitor of subtilisin Amylosacchariticus (10).
Matsubara and Nishimura (12) showed that both the esteratic and
proteolytic activities of subtilisin B P N were inhibited simultaneously
by DFP. Furthermore, Matsubara (54) crystallized the DIP derivative
of subtilisin BPN' and showed that one mole of the inhibitor was bound
to a serine residue per mole of protein. Similar results had been obtained
earlier with subtilisin Carlsberg by Ottesen and Schellman (I?), who
also showed that the reaction with D F P was rapid, requiring about 10
min for 50% inactivation at p H 7.6.
Sanger and Shaw (38)isolated a peptide containing the reactive serine
after partial acid hydrolysis of 32P-DIP-labeled subtilisin Novo. They
showed that the sequence around the serine residue Thr-SerP"-Met-Ala
differed from the Asp-SerP"-Gly sequence found in several mammalian
proteinases sensitive to DFP (55-58). This sequence was extended by
Noller and Bernhard (59) using a specific chromophoric acylating agent,
furylacryloylimidazole, to label the reactive serine residue of subtilisins
BPN' and Novo. After enzymic digestion, a furylacrylyl peptide was
isolated whose sequence was determined as Asn-Gly-ThrSer"-Met.
The determination of the complete sequence by Smith et al. (16) (Fig. 1 )
demonstrated that the active Ser 221 is located in a long sequence of
constant residues from 218 through 240, except for the threonine-serine
substitution a t residue 224 in subtilisin Amylosacchariticus (Fig. 2) (24).
Advantage has been taken of this single highly reactive serine to de-
velop a titration method with the chromophoric acylating agent N -
trans-cinnamoylimidazole to determine the operational normality of sub-
tilisin solutions (60).
1. Serine Sequence in Other Proteinases

It is interesting that some proteinases from microorganisms, notably
those from Streptomyces griseus (81) and Sorangium sp. (62) possess
54. H. Matsubara, J . Biochem. ( T o k y o ) 46, 107 (1959).
55. G. H. Dixon, D. L. Kauffman, and H. Neurath, JACS 80, 1260 (1958).
56. N. K. Schaffer, L. Simet, S. Harshman, R. R. Engle, and R. W. Drisko.
JBC 225, 197 (1957).
57. B. S. Hartley, M. A. Naughton, and F. Sanger, BBA 34, 243 (1959).
58. J. A. Gladner and K. Laki, JACS 80, 1263 (1958).
59. H. F. Noller and 8. A. Bernhard, Bioche7nistry 4, 1118 (1985).
60. M. L. Bender, M. L. Bequk-Canthn, R. L. Blakeley, L. J. Brubacher. J.
Feder, C. R. Gunter, F. J. Kkzdy, J. V. Killheffer, Jr., T. H. Marsliall. C. C.
Miller, R. W. Roeske, and J. K. Stoops, JACS 88, 5890 (1966).
61. S. Wiihlby and L. Engstriim. BBA 151,402 (1968).
62. L. B. Smillie and D. R. Whitaker, JACS 89, 3350 (1967).
16. SUBTILISINS 577
the same sequence around the reactive serine as the mammalian protein-
ases, whereas proteinases from other organisms and plants resemble the
subtilisins ; e.g., those from Aspergillus oryzae ( 6 3 ) , Aspergillus flavus
(64), and a caseinase from French beans ( 6 5 ) . Indeed, other sequences
have been reported for other DFP-sensitive proteinases, e.g., a proteinase
from baker's yeast and phaseolin of French beans possess the sequence
Glu-Ser"-Val (65) and a proteinase from Arthrobacter has the sequence
Ser-Ser"-Gly (66).Table V shows the presently known sequences around
the reactive serine residue of various DFP-sensitive proteinases.
In view of the substantial variations in these sequences, it would
appear that the residues in the sequences near the active serine residue
may have little influence on the reaction mechanism, the reactivity of
the serine hydroxyl, or in determining the specificity of the enzyme;
however, these sequences are useful in classifying the various types of
enzymes.
2. Conversion of Active Serine t o Cysteine

The conversion of the reactive serine residue in subtilisin Novo to
cysteine has been reported by PolgBr and Bender (70)and by Neet and
TABLE V
AROUND REACTIVE
SEQUENCES SERINERESIDUES"
Source Sequenceb
Various mammalian enzymes Asp-Ser'-Gl y

Sorangium sp.
Streptomyces griseus
Pseudocholine esterase Glu-Ser'-Alac
Subtilisins Thr-Ser'-Met-Ala
Caseinase (beans)
Aspergillus
Baker's yeast Glu-Ser'-Val
Phaseolin (beans)
Arthrobacter Ser-Ser'-Gl y
a Reactive serine residues indicated by an asterisk.
* See text for references.
c For pseudocholine esterase see Jansz p t al. (67).The same sequence has also been re-
ported for acetylcholine esterase (68) and for liver aliesterase (69).
63. D. C. Shaw, Ph. D. Dissertation, University of Cambridge (1962).

64. 0. Mike;, J . Turkovii, N. B. Toan, and F. Sorm. BBA 178, 112 (1969).
65. D. C. Shaw and J. R. E. Wells, BJ 104,5c (1967).
66. S. Wahlby, BBA 151, 409 (1958).
67. H. S. Jansz, D. Brone, and M.G . P. J. Warringa, BBA 34, 573 (1959).
Koshland ( 7 1 ) . In both cases the proteinase was reacted first with

phenylmethyl sulfonyl fluoride to convert the serine to a phenylmethyl
sulfonyl derivative (72) and completely inactivate the enzyme. This
group was then displaced by treatment with thiolacetate a t pH 5.25.5
and the thiolester thus formed was allowed to decompose spontaneously
to form thiolsubtilisin as shown by Eq. (1):
+ CaHGHISOaF
Subtilisin-OH subtilisin-O-SO&HzCeHs
- HF I 0
1+
-
-CaHEHnSOr- CHd!!-SH
( 1)
e
0
Subtilisin-SH
+ HIO subtilisin-& -CHs
- CHGOz-
Titration with p-mercuribenzoate (7.9) or 5,5’-dithiobis-2-nitrobenzoic
acid ( 7 4 ) , as well as amino acid analysis, indicated the formation of
0.7-0.9 mole of cysteine per mole subtilisin. No other changes could be
detected by sedimentation analysis, starch gel electrophoresis, fluores-
cence spectroscopy, ultraviolet absorption, or tryptophan reactivity.
However, a small change in the optical rotatory dispersion pattern was
observed (76) .
Study of thiolsubtilisin showed that it had about 33% of the initial
activity of native subtilisin toward p-nitrophenylacetate ( 7 1 ) .Thiolsub-
tilisin also hydrolyzes N-trans-cinnamoylimidazole (76) and the p-ni-
trophenyl esters of several amino acid derivatives although at drastically
reduced rates compared to the native enzyme (75). No activity has
been detected for the thiol enzyme against other ester, amide, or peptide
substrates of subtilisin.
Several findings suggest that the reactions catalyzed by thiolsubtilisin
may be enzymic in character: (1). The intermediate in the hydrolysis
of N-trans-cinnamoylimidazole has the spectral characteristic of a thiol
enzyme ; (2) the hydrolysis of p-nitrophenylacetate follows Michaelis-
Menten kinetics; (3) the hydrolysis of the p-nitrophenyl esters is inhib-
68. D. C. Shaw, unpublished work (1963), cited by F. Sanger, Proc. Chem. SOC.
p. 76 (1963).
69. H. S. Jansz, C. H. Posthumus, and J. A. Cohen, BBA 33,396 (1959).
70. L. PolgBr and M. L. Bender, JACS 88,3153 (1966).
71. K. E. Neet and D. E. Koshland, Jr., Proc. Natl. Acad. Sci. U . S. 56, 1606
( 1966).
72. D. E. Fahrney and A. M. Gold, JACS 85,997 (1963).
73. P. D. Boyer, JACS 76,4331 (1954).
74. G. L. Ellman, ABB 82, 70 (1959).
75. K. E. Neet, A. Nanci, and D. E. Koshland, Jr., JBC 243,6392 (1968).
76. L. Polgtir and M. L. Bender, Biochemistry 6,610 (1967).
16. SUBTILISINS 579
ited by p-mercuribenzoate, whereas this hydrolysis catalyzed by native

subtilisin is not; and (4) the rate of deacylation of cinnamoyl-thiol-
subtilisin and the koat of the p-nitrophenylacetate hydrolysis are de-
pendent, on a basic group with pK;, of about 7.2 (70, 7 6 ) .
These arguments are not completely convincing, however, inasmuch
as there is complete loss of activity of thiolsubtilisin toward normal
ester, amide, and peptide substrates. Furthermore, p-nitrophenyl esters
react rapidly with cysteine itself and can ucylate cysteine residues of
proteins noiienzymically. Although the acylation and deacylation of
thiolsubtilisin are somewhat inorc rapid than with cysteine, the effect
is less impressive than the total lack of activity toward other ester and
amide substrates. Neet et al. (71, 7 5 ) have ascribed this to the changes
in the geometry a t the active site clue to the increased radius and the
change in bond angles of the sulfur atom a s compared to oxygen.
With the more specific ester substrate A'-carbobenzyloxyglycinate
p-nitrophenyl ester, Polgbr and Bender ( 7 7 ) found thiolsubtilisin to be
three orders of magnitude lower in activity than native subtilisin Novo.
They showed also that the thiolenzyme has different chromatographic
properties on carboxymethyl cellulose than the native enzyme. These
results suggest that there may be a subtle change in the conformation
of the active site of the thiolenzyme, and these authors argued on kinetic
grounds for a hydrogen bond between the sulfhydryl group and the
reactive histidine residue in thiolsubtilisin, whereas a similar hydrogen
bond betwerii the serine hydroxyl aiid imidazole does not exist in native
subtilisin. The hydrogen bond formation in thiolsubtilisin also could
cause the loss of one positive charge accounting for the chromatographic
differences between native and thiolsubtilisin. I t is of interest that during
chromatography of native subtilisin Novo another, as yet unidentified,
proteinase impurity was observed. This proteinase, which is presumably
present in all preparations of subtilisin NOVO,could have caused some
of the problems in the sulfhydryl stoichiometry originally observed in
the preparation of thiolsubtilisin.
P o l g h (78) has reported that conversion of the reactive serine to
cysteine in subtilisin Carlsberg produces similar changes in the catalytic
properties and chromatographic behavior as in subtilisin Novo.
The bifunctional alkylating reagent, 1,3-dibromoacetone, which reacts
first with the active cysteine in papain, ficiii, and stem bromelain (79,
80) and then reacts intramolecularly with an active histidine residue,
77. L. Polgir and M . L. Bender, Biochemistry 8, 136 (1969).
78. I,. Polgi\r, Acln Biochim. Biophys. Acrid. Sci. Hung. 3, 397 (1968).
79. S. S. Husain and G. Lowve, BJ 108, 855 and 861 (1988).
80. S. S. Husain and G. Lowe, BJ 110, 53 (1968).
580 F. S. MARKLAND, J R . , AND E. L. SMITH
reacts only with the cysteine in thiolsubtilisin (80)and does not bridge
to the active His 64 (46) that, a t least in subtilisin BPN’, is close to
the active serine ( 4 7 ) .
B. HISTIDINE
The involvement of histidine in the activity of proteolytic enzymes

has been postulated for some years; for the early literature, see, for
example, the reviews by Barnard and Stein (81) and by Dixon et al.
(82)*
The first suggestion of histidine involvement in the active site of
subtilisin was provided by photooxidation studies of Oosterbaan and
Cohen (83). Subsequently, during studies on the sequence around the
reactive serine of subtilisin with the chromophoric acylating agent furyl-
2-acryloylimidazole, Noller and Bernhard (69) suggested that a histidine
residue may be acylated prior to acylation of the reactive serine. They
inferred the presence of a catalytically active histidine residue in spatial
proximity to the serine. The native furylacryloyl enzyme spectrum and
the deacylation rates observed for the native acyl enzyme were different
from those expected for the 0-acyl serine derivative, and were more
similar to those of an acyl histidine derivat‘ve (84,85).
Kinetic evidence for the involvement of histidine was provided by
Glazer (39) who showed that protonation of a group with a pK’ of 6.58
resulted in the inactivation of subtilisin Carlsberg. The heat of ioniza-
tion of this group, 7.3 kcal/mole a t O”, also fell within the expected range
for a histidine residue. Similar results were obtained with subtilisins
BPN’ and Novo (Fig. 4) (86, 87). Svendsen (88) showed that the rate of
inactivation of subtilisin Novo by cyanate is influenccd by a group with
a pK around 7.7. He suggested that a histidine residue might act as
the primary receptor from which the carbamyl group would then be
transferred to the serine residue a t the active site.
Although the above evidence suggested the involvement of a histidine
81. E. A. Barnard and W. D. Stein, Advan. Enzymol. 20,51 (1958).

82. G. H. Dixon, H. Neurath, and J. PechBre, Ann. Rev. Biochem. 27, 489 (1958).
83. R. A. Oosterbaan and J. A. Cohen. in “Structure and Activity of Enzymes.”
(T.W. Goodmin, J. I. Harris, and B. S. Hartley, eds.), p. 87. Sew York, 1964.
84. S. A. Bernhard, S. J. Lau, and H. Noller, Biochemistry 4, 1108 (1965).
85. S. Bernhard, Z. Grdinic, H. Noller. and N. Shaltiel, Proc. Natl. Acad. Sci
U.S. 52, 1489 (1984).
86. H. Lineweaver and D. Burk. JACS 56,658 (1934).
87. B. Myers and A. N. Glazer, unpublished data (1970).
88. I. Svendsen, Compt. Rend. Trav. Lab. (’arkberg 36, 235 (1967).
16. SUBTILISINS 581
a
0 700 -@)
60 64 68 72 76 80
PH
FIG.4. Variation of VmaTwith pH for (A) the hydrolysis of benzoyl-L-arginine
ethyl ester by subtilisin Novo and for (B) the hydrolysis of tosyl-L-arginine methyl
ester by subtilisin Amylosacchariticus. Reactions were at 37” with automatic addi-
tion of 0.02 N base in a pH stat. Initial velocities were determined from the first
5% of the hydrolysis and VmaXwas obtained from the Lineweaver-Burk plot (86).
A molecular weight of 27,600 was assumed. Data were taken from Myers and Glazer
(87). Results similar to those with subtilisin Novo were also obtained with sub-
tilisins BPN’ and Carlsberg (39).
residue, direct chemical evidence was lacking. Specific reagents used for
labeling the histidine residues of trypsin (89) and chymotrypsin (90)
were found to be ineffective in inactivating the subtilisins ( 1 6 ) . Further-
more, other alkylating agents such as bromopyruvate, bromoacetate,
iodoacetate, and iodoacetamide were without effect over a broad range
of pH (24, N u ) .
Shaw and Ruscica (91) synthesized a highly reactive phenylalanine
derivative similar to the chymotrypsin inhibitor, tosyl-L-phenylalanine
chloromethyl ketone. The new reagent, benzyloxycarbonyl-L-phenylala-
nine bromomethyl ketone (ZPBK), was shown to react. stoichiometrically
89. E. Shaw, M. Mares-Guia, and W. Cohen, Biochemistry 4, B19 (1965).
90. G. Schoellmann and E. Shaw, Biochemktry 2, 252 (1963).
90a. E.L. Smith, F. S. Markland, and A. N. Glazer, in “Structure-Function Re-
lationships of Proteolytic Enzymes” (P. Desnuelle. H. Neurath, and M. Ottesen,
eds.), p. 160. New York, 1970.
91. E.Shaw and J. Ruscica, JBC 243, 6312 (1988).
with subtilisin B P N with a concomitant loss of activity. The reaction

was specific and resulted in the loss of a single histidine residue. Sub-
sequent studies by Markland e t al. (@), using tritiated ZPBK, led to
the isolation and identification of a covalently labeled peptide from
subtilisin Carlsberg. It was thus unequivocally demonstrated that His
64 is the active histidine. Similarly, a peptide was isolated from subtilisin
BPN' which contained the labeled histidine residue and was also consis-
tent with its being His 64.
Although His 64 is far removed in linear sequence from the active
Ser 221, the X-ray analysis of subtilisin BPN' showed these residues to
be in close proximity to one another ( 4 7 ) . This situation is strikingly
similar to that in a-chymotrypsin and trypsin where the reactive histi-
dine residues, 57 and 46, respectively, are also close to the amino-terminal
ends and remote from the active serine residues, 195 and 183, respec-
tively (32).
Although the reactive serine residue in the subtilisins is in an invariant
or nearly invariant sequence, this is not the case for the reactive histi-
dine. Residue 63 on the amino-terminal side of histidine is glycine for the
Carlsberg enzyme and serine for B P N (see Table VI which also shows
for comparison the sequences around the reactive histidine residues in
TABLE VI
AROUND REACTIVE
SEQUENCE HISTIDINE
RESIDUES"
Enzyme Sequenceb
Chymotrypsins A and B (bovine) Val-Thr-Ma-Ma-His'-Cy s G ly-Val-Thr

57
Elastase (porcine) Met-T hr-Ala-Ma-His'-Cys-Val-Asp-Arg
57
Trypsin (bovine) Val-Ser-Ala-Ala-His'-Lys-Tyr-Lys-Ser
57
Subtilisin Carlsberg Asp-Gly-Asn-Gly-His'-Gly-Tbr-His-Val
64
Subtilisiri BPN' Asp-Asp-Asn-Ser-His*-Gly-T hr-His-Val
64
~~
a The residue numbers are under the sequence; for the mammalian proteinases the
numbering system is that of chymotrypsinogen A. The reactive histidine residue is
marked by an asterisk.
b For review of references for the pancreatic proteinases, see Smillie and Hartley (92)
and for the subtilisins, see Markland el al. (46).
92. L. B. Smillie and B. S. Hartley, BJ 101, 232 (1966).

16. SUBTILISINS 583
several mammalian proteinases) (46,92). This single amino acid replace-

ment may account in part for the greater activity of subtilisin Carlsberg
and some of the small differences in specificity exhibited by these two
enzymes.
Subtilisin Amylosacchariticus has a lower specific activity for syn-
thetic ester substrates than the other two subtilisins and is also much
less reactive with ZPBK (Fig. 5 ) (24). Since this enzyme also has
distinctive kinetic properties, we would expect these differences in
reactivity to be reflected in the sequence and conformation of this
enzyme.
Morihara and Oka (92a) have syntlietized the chloromethyl ketone
derivatives of two peptide substrates of subtilisin BPN' (carbobenzoxy-L-
Ala-Gly-L-Phe chloromethyl ketone and carbobenzoxy-L-Ala-L-Phechlo-
romethyl ketone) and have shown that these compounds react much
faster with the enzyme than ZPBK. The reaction was stoichiometric and
Time (hours)
FIG. 5. Comparison of the reactivity of subtilisins Carlsberg and Amylosac-

chariticus with tritiated ZPBK (24). The reaction waa carried out at pH 6.8 in a
volume of 6.0 ml containing (final concentration): 33% dioxane 0.093M trisqC1,
0.0093 M CaC12,12.6 pmoles ZPBK and 029 pmole protein. At various times aliquots
wrre removed to assay the esterase activity on acetyl-L-tyrosine ethyl ester by the
method of Glazer (39). Control samples contained dioxane without ZPBK. (A)
Subtilisin Carlsberg control ; (B) subtilisin Carlsberg plus 'HZPBK; ( C ) subtilisin
Amylosacchariticus control, and (D) subtilisin Amylosacchariticus plus SH-ZPBK.
92a. K. Morihara and T. Oka, ABB 138,526 (1970).

inactivation was partly prevented by the presence of hydrocinnamic acid.

Amino acid analysis indicated that a histidine residue had been alkylated.
VI. Substrate Specificity and Enzymic Properties
Since the discovery of the subtilisins, it has become evident that these
enzymes manifest a broad specificity as proteinases. During purification
of the Carlsberg enzyme esterase activity was observed on methyl
butyrate, as well as proteinase activity on casein, hemoglobin, oval-
bumin, and gelatin. The pH optimum for casein digestion was in the
range from p H 10 to 11 ( 7 ) . Graae (93) showed that the esterase and
proteinase activity was associated with the same enzyme.
Okunuki et al. (94, 95) reported that subtilisin BPN’ also hydrolyzed
a variety of proteins, including casein, hemoglobin, gelatin, and oxidized
lysozyme. Cleavage appeared to occur mainly a t the NH,- and COOH-
terminal sides of neutral and acidic amino acid residues ( 9 4 ) . In addi-
tion, subtilisin BPN’ was shown to have esterase activity with a pH
optimum around 8.5-9.5 (11).
A. PROTEIN SUBSTRATES
AND PEPTIDE
Tuppy ( 9 6 ) ,Haugaard and Haugaard (97), and Meedom (98) studied

the hydrolysis of the insulin chains by subtilisin and found extensiw
cleavage. It was evident that subtilisin hydrolyzed a great variety OJ
peptide bonds, e.g., Meedom found that 20 of the 49 peptide bondr
present in porcine insulin were cleaved. Furthermore, hydrolysis occurrec
not only a t internal peptide bonds but also a t terminal bonds to yielc
free amino acids. Early work on the specificity of subtilisins with pro.
tein substrates was reviewed by Hagihara (1).
Digestion of oxytocin by subtilisin resulted in cleavage a t the car.
boxy-terminal end of glutamine and leucine (99) which is consisten
93. J. Graae, Actu Chem. Scand. 8,350 (1954).
94. K. Okunuki, H. Matsubara, S. Nishimura, and B. Hagihara, J. Biochem
(Tokyo) 43, 857 (1956).
95. B. Hagihara, M. Nakai, H. Matsubara, T. Komaki, T. Yonetani, and K
Okunuki, J . Biochem. (Tokyo) 45, 305 (1958).
96. H.Tuppy, Monatsh. Chem. 84, 996 (1953)
97 E. S. Haugaard and N. Haugaard, Compt. Rend. Trav. Lab. Carlsberg, Se
Chim. 29, 350 (1955).
98. B. Meedom, Compt. Rend. Trav. Lab. Curlsberg, Ser. Chim. 29, 403 (1955
99. H. Tuppy, BBA 11,499 (1953).
16. SUBTILISINS 585
with the specificity for aromatic or apolar residues obtained with small
ester and amide substrates (@,100).
Reinvestigation of the subtilisin-catalyzed cleavage of insulin under
restricted conditions yielded more definitive information concerning the
specificity of subtilisin. Morihara and Tsuzuki (100) found that after
digestion of the oxidized insulin B chain for 2-3 hr with 0.1% by weight
of subtilisin B P N a t room temperature and pH 9 cleavage occurred
only a t peptide bonds 6 5 (Gln-His) , 9-10 (Ser-His), 15-16 (Leu-Tyr),
16-17 (Tyr-Leu), and 25-26 ( P h e T y r ) . This is consistent with the
findings of these authors that hydrolysis of small ester and amide sub-
strates such as acetyl-X-ethyl ester and carbobenzoxy (Cbz)-Gly-X-
NH, occurs a t the carboxy-terminal side of residue X (where X rep-
resents aromatic or apolar residues such as L-tyrosine, L-phenylalanine,
and L-leucine) . Earlier, Ottesen and co-workers (101) compared the
specificities of subtilisins Carlsberg and Novo after hydrolysis of the B
chain of oxidized insulin a t 30' and pH 8 for 2 hr a t very low enzyme-
to-substrate ratios. Digestion by subtilisin Carlsberg a t an enzyme to
substrate ratio of 1 :3960 produced 12 peaks after separation of the pro-
ducts of digestion on Dowex 50-X4. The two major peptides, however,
obtained in about 50% yields were composed of residues 1-15 and 16-30,
indicating that the major point of cleavage was a t the Leu-Tyr bond
(residue 15-16) of the oxidized insulin B chain. When the enzyme con-
centration was increased, peptide segments 1-15 and 16-30 disappeared
and were replaced by fragments representing hydrolysis a t residues
4-5 (Gln-His), 9-10 (Ser-His), and 11-12 (Leu-Val) for fragment
1-15, and a t residues 16-17 (Tyr-Leu), 17-18 (Leu-Val), and 26-27
(Tyr-Thr) for fragment 16-30.
Similar results were obtained with subtilisin Novo, but the relative
rates of hydrolyses were different. Thus peptide bonds 9-10 (Ser-His)
and 26-!27 (Tyr-Thr) were hydrolyzed faster by subtilisin Novo than
by subtilisin Carlsberg while the opposite was the case with bonds 4.5
(Gln-His) and 11-12 (Leu-Val) .
Ottesen and gstergaard (102)also observed slight differences between
subtilisins Novo and Carlsberg in the conversion of ovalbumin to plak-
albumin. At an enzyme-to-protein ratio of one to l0,OOO a t 30" and pH
8 both subtilisins Novo and BPN' acted in identical fashion on oval-
bumin, as judged by the solubility of the plakalbumin formed, but
100. K. Morihara and H. Tsuruki, ABB 129, 620 (1989).
101. J. T. Johansen, M. Ottesen, I. Svendsen, and G. Wybrandt, Compt. Rend.
Truv. Lab. Curlsberg 36, 365 (1968).
102. M. Ottesen and B. Ostergaard, Compt. Rend. Trav. Lab. Carlsberg 34, 187
(1964).
differently from subtilisin Carlsberg. Nonetheless, all three proteinases

liberate the same major peptide fragment indicating that the primary
site of attack is identical, although the composition of the larger peptide
fragments released by the Novo enzyme appeared more complicated than
those liberated by subtilisin Carlsberg indicating some differences in
specificity.
The well-known action of subtilisin Carlsberg on ribonuclease, under
controlled conditions, involves the hydrolysis of a single peptide bond
producing the S protein and S peptide (103, 104). Exhaustive digestion
of ribonuclease by subtilisin Carlsberg, on the other hand, produces
cleavage at about 25% of the peptide bonds in the native protein (106,
106). Subtilisin BPN' also cleaves ribonuclease forming the active RNase
S derivative (107). However, on closer examination it was found that
whereas the major (84%) cleavage point with subtilisin Carlsberg was
between residues 20 and 21 (Ala-Ser) , subtilisin BPN' produced peptide
fragments indicating substantial cleavage at both bonds 20-21 (Ala-
Ser) and 21-22 (Ser-Ser) (108, 109). In fact, it is possible that the initial
site of subtilisin B P N cleavage is at bond 21-22 and that only during
further digestion is the COOH-terminal serine (residue 21 of the S pep-
tide) partially lost. This would then be a clear-cut difference in specificity
inasmuch as B P N favors cleavage a t bond 21-22 whereas subtilisin
Carlsberg cleaves preferentially at bond 20-21.
B. SYNTHETIC
SUBSTRATES
The subtilisins show low specificity when tested on a wide variety of

benzoyl- or acetyl-L-amino acid esters. In comparing subtilisins NOVO,
BPN' and Carlsberg, Ottesen and Spector ( 9 ) and Hunt and Ottesen
(288) observed that benzoyl-L-tyrosine ethyl ester was a much better
substrate than benzoyl-L-leucine ethyl ester for subtilisin BPN' (and
Novo) but both were about the same for subtilisin Carlsberg. For both
substrates subtilisin Carlsberg was more active than Novo and B P N
103. F. M. Richards, Compt. Rend. Trav. Lab. Carlsberg, Ser. Chim. 29, 329
( 1955).
104. F. M. Richards and P. J. Vithayathil, JBC 234,1459 (1959).
105. F. M. Richards, Compt. Rend. Trav. Lab. Carlsberg, Ser. Chim. 29, 322
(1955).
106. M. Ottesen and M. Szhkely, Compt. .Rend. Trav. Lab. Carlsberg 32, 319
(1962).
107. G. Gordillo, P. J. Vithayathil, and F. M. Richards, Yale J . Bwl. Med. 34,
582 (1962).
108. M. 5. Doscher and C. H. W. Hirs, Biochemistry 6, 304 (1987).
109. E. Gross and B. Witkop, Biochemistry 6, 745 (1967).
16. SUBTILISINS 587
whereas the last two were kinetically indistinguishable. By comparison

chymotrypsin was 50 times more active on benzoyl-L-tyrosine ethyl
ester than subtilisin Carlsberg.
Glazer (S9),Bare1 and Glazer (@), and Myers and Glazer (87)have
determined kinetic parameters for the hydrolysis of a number of N-
acetylamino acid esters by subtilisins NOVO,Amylosacchariticus, and
Carlsberg. The data are given in Table VII. All of the subtilisins show a
markedly higher activity on the esters of aromatic amino acids than on
those of aliphatic ones. For subtilisin Carlsberg V,, values were higher
than for the other enzymes with N-acetyl aromatic amino acid esters;
subtilisin Amylosacchariticus was the least active. This suggests that
the deacylation rate is faster for subtilisin Carlsberg than for the other
two subtilisins. This has been verified by the finding that the deacylation
rate of N-trans-cinnamoyl subtilisin Carlsberg is significantly higher
than the corresponding rate for subtilisin Novo (40). Also the kinetics
of the deacylation of indoleacryloyl subtilisins Novo and Carlsberg has
been studied as a function of pH (110). Essentially the same results were
obtained : the deacylation rate for subtilisin Carlsberg was substantially
higher than for the Novo enzyme, and both enzymes showed dependency
on a group with pK,,, about 8. The deacylation rate constant for the
indoleacryloyl derivatives of a-chymotrypsin, trypsin, and subtilisin
Novo were all very similar but much smaller than the corresponding
constant for subtilisin Carlsberg. The large differences in the deacylation
rate constants were maintained even when using the iodo, nitro, succinyl,
and glutaryl subtilisin Carlsberg derivatives and the nitro subtilisin
Novo derivative. It would appear, therefore, that the lysyl residues and
the accessible tyrosine residues are not involved in the hydrolysis of
indoleacryloyl subtilisin Carlsberg. These data also support the findings,
which are discussed below, that with low molecular weight substrates
modifications of the subtilisins produced no .change in enzymic activity
(110).
From both the kinetic data (Table VII) and the amino acid sequence
subtilisin Amylosacchariticus closely resembles the B P N and Novo
enzymes. Nevertheless, as shown in Fig. 4, V,,, for Amylosacchariticus
is independent of pH over the range 6-8 with both tosyl-L-arginine
methyl ester and acetyl-L-tyrosine ethyl ester as substrates (87),
whereas, over this same pH range, both subtilisins Novo and Carlsberg
show a sigmoid dependence of VmaXon pH (39).Apparently, a different
rate controlling step dominates the kinetics in the Amylosacchariticus
enzyme.
110. J. T.Johansen, R. W.A. Oliver, and I. Svendsen, Compt. Rend. Trav. Lab.
Carkrberg 37, 87 (1969).
TABLE V I I
KINETICCONSTANTS
FOR SUBTILISIN-CATALYZED
HYDEOLYSIS ACID ESTERS~
OF N-ACETYUMINO
Subtilisin
Amylosacchariticu9 Novae Carlsberg BPNtd
Substrate
N-Acetyl-btyrosine ethyl ester 0.146 548 0.07 731 0.09 1316 0.0222 383.3
N-Acetyl-ctyrosine methyl ester 0.046 281 0.09 1560 0.07 1930
N-Acetyl-btryptophan methyl ester 0.031 149 0.09 415 0.05 820
N-Acetyl-cphenylalanine methyl ester 0.044 93.9 0.06 415 0.03 765
N-Benzoyl-carginine ethyl ester' 0.012 19.8 0. 007b 3.9 0.007 16.1 0.010 3.1
N-Tosyl-barginine methyl ester' 0.042 17.7 0.07 15.5 0.044 68.6
N-Acetyl-bvaline methyl ester 0.28 28 0.19 23 0 F
N-Acetylglycine ethyl ester 0 m
N-Acetyl-calanine methyl ester 0.123 72.0
N-Acetyl-bnorvaline ethyl ester 0
N-Acetyl-bleucine methyl ester 0.0666 57.5
N-Acetyl-cphenylalanine ethyl ester 0.0166 30.6
N-Acetyl-btryptophan ethyl ester 0.0238 35.8
N-Acetyl-clysine methyl ester 0.0909 47.4
4
pl
a The reaction mixture contained, initially, 0.0025 to 0.25 M substrate in 5.0 ml of 0.1 M KC1 containing 8% dioxane of volume.
The suhtilisin concentration was 0.0028 to 0.4 mg/ml. Titrations were performed a t pH 8.0, 37", with 0.02 M base as titrant.
b Data from Myers and Glazer (87).
c Data from Glazer (39) and Bare1 and Glazer (40).
Data from Morihara and Tsuzuki (100).Conditions differed as follows: measurements at 30Oand pH 7.5 using 0.05 N NaOH as titrant.
Reaction mixtures contained initially from 0.2 to 35 mM substrate in 5.0 ml of 0.1 M KCl containing 4% ethanol. Subtilisin concentra-
tion was adjusted to the range where the rate of reaction was proportional to the enzyme concentration.
0 Calculated per mole of enzyme, based on a molecular weight of 27,600.
f No dioxane in reaction mixture.

16. SUBTILISINS 589
The finding (39) that certain aromatic compounds, e.g., phenol, indole,
hydrocinnamate, and indolepropionate, are competitive inhibitors of the
hydrolysis of N-acetyl-L-tyrosine ethyl ester but are noncompetitive in-
hibitors with N-a-benzoyl-L-arginine ethyl ester suggests that these two
ester substrates are bound in a somewhat different manner at the active
sites. Both modes of binding obviously lead to productive complexes, a
fact which may in part explain the broad range of specificity of the
subtilisins. Thus different substrates have different productive modes of
binding and the subtilisins are therefore able to cleave peptide bonds
in a wide variety of natural and synthetic substrates.
Although subtilisin Carlsberg has a higher V,,, for the acetylamino
acid esters than either subtilisins Novo or Amylosacchariticus, the K,
values for the substrates (Table VII) and the KI values for the inhibitors
(Table VIII) are similar for all of these enzymes indicating that, despite
differences in amino acid sequences they possess similar substrate binding
sites. It has been observed (39), however, that subtilisin Carlsberg has
higher V,,, values with the N-acetylamino acid esters than subtilisin
NOVO,but that both have similar V,,, values with the free amino acid
esters (40). This suggests that the binding site of Carlsberg is less polar
than that of the Novo enzyme and thus more sensitive to the polar
a-amino group in the free amino acid esters (40).
The broad specificity exhibited by the subtilisins is in marked contrast
to the high degree of specificity shown by trypsin and chymotrypsin
toward synthetic substrates (111). Furthermore, the subtilisins have
TABLE VIII
COMPETITIVE
INHIBITORS
OF HYDROLYSES OF ACETYL-L-TYROSINE
&HYL
ESTERBY SUBTILISIN“
Inhibitor Carlsberg Novo BPN’

Inhibit09 conc. (M) PH (MI (MI (MI
Indole 0.00625 7.5 0.05 0.05 0.03
Phenol 0.05 7.4 0.10 0.11 0.10
Hydrocinnamate 0.025 8.0 0.14 0.30 0.34
Methyl butyrate 0.088 7.0 0.17 0.17 0.21
The compounds listed in the table were tested as competitive inhibitors of subtilisin-
catalyzed hydrolysis of acetyl-btyrosine ethyl ester in 0.1 M KCI containing8% dioxane
by volume at 37” and at t,he pH and inhibitor concentration indicated.
* Data from Glazer (39).
111. N . M. Green and H. Neurath, in “The Proteins” (H. Neurath and K.
Bailey, eds.), 1st ed., Vol. 2, Part B, p. 1057. Academic Preas, New York, 1954.
much higher K,,, values with their best ester substrates than do chymo-
trypsin and t.rypsin and also act extremely slowly on acetyl-L-amino
acid amides (39, 100). It would appear that high K , values for simple
ester substrates may be associated with proteinases of low specificity
such as papain and ficin (112) as well as various bacterial and fungal
enzymes (100).
Morihara (113) and Morihara and Tsuzuki (100) have studied the
action on synthetic substrates of a variety of alkaline and neutral pro-
teinases from various bacteria and molds. The neutral proteinases hydro-
lyze mainly at peptide bonds involving the amino group of hydrophobic
amino acid residues (113,114), whereas the alkaline proteinases appear
to hydrolyze mainly a t peptide bonds linking the carboxyl group of
hydrophobic amino acid residues. The data in Table I X indicate that
subtilisin B P N (as do the other alkaline proteinases studied) hydrolyzes
synthetic substrates of the type Cbz-Gly-X-NH, where X is Ala, Leu,
Phe, Tyr, Ser, and Trp (the last two show less extensive hydrolysis).
Benzoyl-Tyr-NH, is hardly hydrolyzed at all by B P N in distinction
to a-chymotrypsin where this amide is hydrolyzed much more rapidly
than Cbz-Gly-Tyr-NH,. To investigate the effect of neighboring groups
on the hydrolysis of synthetic substrates a variety of leucine derivatives
was studied (Table I X ) . The hydrolysis of Cbz-Gly-Leu-NH, is greatly
reduced when the Cbz group is replaced by acetyl and there is almost
no hydrolysis when the amino group is unsubstituted. When L-leucine
is replaced by D-leucine there is no hydrolysis. With various acylamino
acid esters, Morihara and Tsuzuki (100) found, as did Glazer (39) and
Bare1 and Glazer (@), that subtilisin B P N has higher activity when
aromatic amino acids are present, but there is also activity with esters
of the basic amino acids and certain neutral aliphatic amino acids (Table
VII) .
Additional work by Morihara et al. (114a) with leucine substrates of
the type Cbz-A- (Gly),,-Leu-NH, and Cbz-Leu- (GIST),,-B (where A or B
are various D or L amino acid residues; n equals 0, 1, and 2 ; and cleavage
occurs a t the COOH-side of the leucyl residue) have shown that hydrol-
ysis was influenced by the three amino acid residues on the NH,-terminal
side and the two amino acids on the COOH-terminal side of the sensitive
bond. Furthermore, these effects on hydrolysis were mainly related tc
catalysis rather than substrate binding.
112. E. L. Smith and J. R. Kimmel, “The Enzymes,” 2nd ed., Vol. 4, p. 133
1960.
113. K. Morihara, BBRC 26, 656 (1967).
114. K. Morihara, H. Tsuzuki, and T. Oka, ABB 123, 572 (1968).
114a. K. Morihara, T. Oka, and H. Tsuzuki, ABB 138,515 (1970).
16. SUBTILISINS 591
TABLE IX
HYDROLYSES SYNTHETICSUBSTRATES
OF VARIOUS BPN'.
BY SUBTILIBIN
-~
Hydrolysisb Hydrolysis*
20 16 20 16
min hr min hr
Substrate (%I (%) Substrate (%) (%I
Cbz-Gly-Gly-NHz 0 Cbz-Gly-Pro-D-Leu-G1y-Pro-oH 0
Cbz-Gly-Ala-NH2 11 >95 Cbz-Gly-Pro-Leu-Gly-OH 20 >95
T T
Cbz-Gly-Ser-NH2 0 22 Cbz-Gly-Pro-Leu-OH 0
t Cbz-Gly-Pro-NHS 0
Cbz-Gly-Val-NHzc <5 Cbz-Gly-PheNHsC 6 70
Cbz-G ly-Ile-N Hp 0 t
Cbz-Gly-Leu-NHZ 18 >95 (Cbz-Gly-Tyr-NHf 32 >95
T t
Cbz-Gl y-D-Leu-NHz 0 Cbz-Gly-TrpNHnc 0 13
Acety1-Gly-Leu-NH? 11 T
T Benzoyl-Tyr-NHn <5
H-Gly-Leu-NHz <5 Benzoyl-Arg-NHz <5
Cbz-Leu-N Hz <5
Cbz-Gly-Pro-Leu-Gly-Pro-OH 37 95
t I
All reactions were performed in 0.1 M tris buffer (pH 8) for 20 min and for 16 hr at
40". The percentage of hydrolysis was determined by ninhydrin analysis. Substrate con-
centration was 4 mM and tha enzyme concentration was 50 pg/ml of reaction mixture.
The arrows show the points of cleavage.
* Data from Morihara and Tsuzuki (100).
The reaction mixture contained 4% methanol because of the low solubility of the
substrate.
It was also found that hydrolysis was inhibited when the terminal
a-amino and a-carboxyl groups of the peptide substrate were unblocked,
if the former group was within four residues on the NH,-terminal side
and the latter group within one residue on the COOH-terminal side of
the cleavage point. Finally, these authors reported that the side chain
specificity a t the residue being cleaved is extremely stringent compared
with those for each of the five amino acid residues surrounding the sen-
sitive residues, and that bulky residues on either side of the sensitive
bond inhibit hydrolysis.
Morihara and Tsuzuki (100) have called the alkaline proteinases
chymotrypticlike in their specificity. However, in comparing the size and
specificity of the active site of a-chymotrypsin and subtilisin BPN' by
using a variety of synthetic peptides, Morihara et al. (116) found con-
115. K. Morihara, T. Oka, and H. Tsuzuki, BBRC 35, 210 (1969).
siderable differences between these two types of proteinases. By design-

ing synthetic peptides so that hydrolysis occurred a t the bond involving
the carboxyl group of L-tyrosine the effects of neighboring residues were
examined. Thus peptides of the type Cbz-A-Tyr-NH,, Cbz-A-Gly-Tyr-
NH,, Cbz-Tyr-B-NH,, and Cbz-Tyr-Gly-B (where A or B are various
amino acid residues) were used where the subsite numbering system is
S4-&-SZ--S,--S:
Cbz-A-Gly-Tyr-NHs
On the assumption that the substrates bind t o the enzyme in such a way
that the CO-NH linkage being hydrolyzed is always the same and that
the amino acid residues occupy adjacent subsites, those toward the
amino end occupying subsites S,, S,, etc. and those toward the carboxyl
end subsites Si, S:, etc., it was found that both subtilisin BPN’ and
a-chymotrypsin have large active sites which can be divided into a t least
five subsites, each accommodating one amino acid residue of the sub-
strate. Furthermore, stereospecificity is present in both enzymes a t all
subsites. Thus for subtilisin BPN’ the rate of hydrolysis of Cbz-L-Ala-
Gly-L-Tyr-NH, is 15.2 pmoles/min/mg enzyme but with the diastereo-
isomer Cbz-D-Ala-Gly-L-Tyr-NH, the corresponding rate of hydrolysis
is only 0.067. Side chain specificity of the subsites indicated that alanine
a t subsite S, greatly promoted the activity of subtilisin B P N when
compared to glycine, histidine, or tyrosine a t this site. Furthermore,
elongation of the peptide chain toward the NHAerminus in subtilisin
BPN’ greatly stimulated the activity as seen by a comparison of the
rate of hydrolysis for Cbz-Gly-Tyr-NHs, 0.098 pmole/minJmg enzyme,
versus the corresponding rate for Cbz-Gly-Gly-Tyr-NH,, 5.00. Chang-
ing the glycine in subsite S3 of the latter peptide to alanine also promotes
the rate of hydrolysis from 5.0 to 15.2 pmoles/min/mg enzyme, indicat-
ing that subsite SB exerts at least some control over the activity of the
enzyme. This effect does not appear to be true for a-chymotrypsin. Sub-
site S: does not appear to have much effect on hydrolysis by subtilisin
BPN’ whereas it does with a-chymotrypsin. Subsite Sg may have more
of a controlling influence on subtilisin BPN’ although the data are
limited.
1. Unusual Ester Substrates

Subtilisin also catalyzes the hydrolysis of some triglycerides, including
tripropionin and tributyrin (116), and the D but not the L isomer of
l-keto-3-carbomethoxy-1,2,3,4-tetrahydroisoquinoline.The enzyme, how-
116. G. Sierra, Can. J . Microbiol. 10, 926 (1964).

16. SUBTILISINS 593
ever, retains its normal stereospecificity when the related open chain
ester iV-benzoyl alanine methyl ester is the substrate, hydrolyzing the
k
L and not he D isomers. Since a-chymotrypsin behaves in an identical
manner wit these substrates, both enzymes have similar specificity with
respect to the configuration of their substrates (117).
C. TRANSESTERIFICATION
AND TRANSPEPTIDATION
The subtilisins also catalyze transesterification reactions (118) al-

though at a somewhat lower rate than chymotrypsin (119) and trypsin
(120).
No transpeptidation was evident with subtilisins Novo or Carlsberg,
but a small amount occurred with subtilisin Amylosacchariticus cata-
lyzed hydrolysis of tetraalanine (87).Additionally, like most of the
proteolytic enzymes the subtilisins have been shown to catalyze aminol-
ysis reactions (40)as shown in Eq. (2) :
LLericine benzyl ester + glycylglycine n-amyl ester
L-Leucine glycylglycine n-amyl ester + benzyl alcohol (2)
D. MECHANISM
OF ACTION OF THE SUBTILISINS
Much has already been said concerning the mechanism of action of

the serine proteinases, in general, and a-chymotrypsin, in particular
(48, 121-124). Interest was generated in the subtilisins when it was
learned that they were inhibited by DFP (7,12, 17, 26).The observa-
tion of acyl-enzyme intermediates during the catalysis of hydrolytic re-
actions (84)by the subtilisins, similar to those observed for a-chymo-
trypsin, has further emphasized the similarities between these and other
serine proteinases. Specific acylating agents such as N-cinnamoylimida-
zole (60)and p-nitrophenylacetate (76)have been used to titrate the
subtilisins during the attainment of the steady state, and a cinnamoyl
enzyme derivative has been isolated (59, 84). As already discussed, the
involvement of serine and histidine in the activity of the subtilisins is
117. H.Dugas. Con. J. Biochem. 47, 985 (1969).

118. A. N.Glazer, JBC 241, 635 (1966).
119. C.E.McDonald and A. K. Balls, JBC 221, 993 (1956).
120. A. N. Glazer. JBC 240, 1135 (1965).
121. M. L. Bender and F. J. KBzdy, Ann. Rev. Biochem. 34,49 (1965).
122. J. H.Wnng, Science 161, 328 (1%).
123. A. Williams, Quurt. Rev. (London) 23, 1 (1969).
124. G.P.Hess. “The Enzymes,” 3rd ed.. Vol. 111, Chapter 7, 1971.
now well documented and a role for Asp 32 has been postulated ( 4 7 )
similar to the involvement of a specific aspartic acid in the mechanism
of a-chymotrypsin and other pancreatic proteinases ( 4 8 ) .
Keizer and Bernhard (125) noted that the protonic equilibrium ac-
companying acylation of both subtilisin Novo and a-chymotrypsin with
N-P- (3-indole) acryloylimidazole is identical, indicating that the in-
volvement of proton diseociable groups in the mechanism of catalysis
must be similar for these two enzymes. Thus, hydrolysis presumably
occurs via acylation ( k L ) and deacylation (Ic,) steps according to
Eq. (3):
EH + RCOX ek-i
kl
EH.RCOX
kg
k-2
ECOR + HX
(3)
ECOR + HnO e IlCOiH + EH
ks
k-,
An interesting observation that was made during the studies with thiol-
subtilisin (Section V,A) may shed sonic light on the general mccha-
nism of the serine proteinases. The high reactivity of the serine hydroxyl
at the active site of serine proteinases has been generally attributed to
hydrogen bonding between this residue and a nearby histidine residue
(122) which in turn is hydrogen bonded to an aspartic acid residue
(47, 48). However, Polg6r and Bcndcr (126, have presented a contrary
view. By measurement of the kinetic parameters it was shown that sub-
tilisins Novo and Carlsberg and their thiol derivatives in the acyl-
enzyme form possess an ionizable group with a pK, of about 7.0 in the
active site. This group, presumably the active histidine residue, also is
present in the unsubstituted enzymes but not in the free thiol derivatives
(76, 7 8 ) . The p l i has been shifted to 6.15 in free thiolsubtilisin Carls-
berg and somewhat lower than pH 5.5 in thiolsubtilisin Novo. To explain
this they postulate that a hydrogen bond is present between the thiol
group and the nearby histidine residue in the free thiol enzymes, whereas
in the serine enzymes, since there was 110 shift in the pK between the free
and aeyl enzymes, it appeared that no hydrogen bond is present. Kinetic
studies with D 2 0 supported the above conclusions. I t is known that the
velocity of reactions with a rate limiting protori transfer decrease to
about one-third the normal rate in D,O. The formation of a hydrogen
bond is coiisidcrctl a partial proton transfer; however, if a hydrogen bond
were formed between the catalytic groups during the formation of acyl
enzyme the rate would riot I)c markedly affected in D,O. The results in-
dicate that there was a D,O effect for deacetylation with p-nitrophenyl-
125. J. Keizcr nnd S. .I.Bcrnhard. B i o c h ~ m i ~ t r5,y 4127 (1966)
126. I,. Polgtir and M. L. Bender. Proc. Natl. Acnd. Sci. U . S. 64, 1335 (1969).
16. SUBTILISINS 595
acetate both for subtilisin and thiolsubtilisin (Carlsberg) indicating a

rate controlling proton transfer in the catalytic step. However, in the
formation of the acetyl enzyme there was only a rate determining proton
transfer in the serine enzyme. The thiolenzyme showed no effect of D,O
on the rate, supporting the suggestion of a hydrogen bond between the
thiol group and the imidazole group. This is contrary to the hypothesis
of Wang (122) who explained the reactivity of the hydroxyl group of
the serine proteinases as resulting from a rigidly and accurately held
hydrogen bond of the serine OH to an imidazole group. Furthermore, he
postulated that the reason for the inefficiency of the thiol enzyme was
that this hydrogen bond was disrupted.
Figure 6 shows the mechanism of formation and hydrolysis of the acyl
enzyme as envisioned by Polgir and Bender (186) which incorporates
features of the mechanism proposed by Wang (199) and by Blow et al.
(48). According to this view, all that is needed to initiate the catalytic
reaction is a favorable collision between the carbonyl carbon atom of the
substrate and the oxygen atom of the serine residue. Bond making, prob-
ably the formation of a tetrahedral intermediate, and proton transfer to
the imidazole proceed in a concerted manner. This step which involves
general base catalysis is followed by a general acid-catalyzed step in
which a proton is transferred from the imidazolium to the leaving group
in a concerted manner with bond breaking. Between the two general
0
Oh
C -I Asp
OdH
FIG.6. Possible mechanism of the formation (a), and the hydrolysis (b) of the
ncyl enzyme in the catalysis by serine proteases; X represents the leaving group.
From Polgir and Bender (126).
catalyses the imidazolium is stabilized by the aspartate-imidazole hy-

drogen bond system.
VII. Chemical Modification Studies
Although the subtilisins show variations in primary structure as well

as in kinetics and specificity, they are very similar in other ways:
( 1 ) They are resistant to many denaturing agents including 6 M
urea, 50% ethanol, and anionic detergents a t room temperature
(26, 27).
(2) These enzymes are rapidly and irreversibly denatured below pH
5 (26).
(3) Aromatic compounds are competitive inhibitors ; this suggests
the presence of a hydrophobic binding site (16, 39).
These and other findings have led to studies aimed a t the specific
modification of the subtilisins in attempts to determine which residues
are in the substrate binding site, which are involved in the acid lability
of the enzyme, and which are otherwise essential for the activity and
stability of the enzyme.
A. LYSINEMODIFICATION
I n the pH region where subtilisin is labile, below pH 5, it was sup-

posed that there may be a drastic net increase in positive charge because
of the protonation of carboxylate ions or of imidazole groups. Thus, the
acid lability might result either from a general charge effect, namely,
disruption of the native structure by electrostatic charge repulsion, or
from a local charge effect whereby protonation of a particular car-
boxylate or imidazole group causes a drastic conformational change. By
succinylating the lysine residues there would be a drastic net change in
the charge of the protein and thus a means would be provided for sepa-
rating a general charge effect from a local charge effect. Succinic anhy-
dride reacted with the <-amino groups of 10-11 of the 11 lysine residues
in subtilisin Novo essentially without affecting the ionization state of the
carboxyl groups or the histidine residues (26); however, as will be dis-
cussed later, several t,yrosine or serine and threonine residues were also
succinylated. Nevertheless, with a t least 10 lysine residues succinylated,
there is a net change in charge of a t least minus 20 and the enzymic ac-
tivity is essentially unchanged with benzoyl-L-tyrosine ethyl ester or
16. SUBTILISINS 597
benzoyl-L-leucine ethyl ester as substrates over a wide pH range. With

the small basic protein clupeine as substrate, a more complex picture was
observed and this will be discussed in Section VI1,C. The succinylated
enzyme appears only slightly less stable than the native enzyme through-
out the entire pH range, and it is homogeneous by both moving boundary
electrophoresis and ultracentrifugation with no evidence for association
or dissociation. Optical rotation measurements, although sparse, sug-
gested no drastic changes in conformation of the succinylated enzyme.
Thus, since succinylation has little effect on catalytic activity or sen-
sitivity to acid pH, it would appear that it is not a general charge effect
which is responsible for the acid lability of the subtilisins, but it is pre-
sumably a result of specific local effects probably involving either car-
boxylate or imidazole groups ( 1 2 6 ~ )Additionally,
. since modification of
10-11 out of 11 lysine residues did little to the enzymic activity on ester
substrates, it appears that lysine residues are not part of the active cen-
ter ; however, their involvement in binding sites for large molecular
weight substrates cannot be excluded. The finding that the lysine resi-
dues are readily accessible for reaction with succinic anhydride suggested
that they are on the surface of the protein ( 2 6 ) .This has been confirmed
by X-ray analysis which indicates that 10 of the 11 lysine residues are
on the surface of subtilisin BPN’ ( 4 7 ) .
Succinylation of subtilisin Carlsberg modifies 6 of the 9 lysine residues
and 14-16 hydroxyl groups of threonine or serine as well (presumably
a similar reaction occurs in subtilisin Novo) (101).
Carbamylation of subtilisin Novo converted 10-11 of the 11 lysines
into homocitrulline residues but almost completely inactivated the en-
zyme as estimated from its proteolytic activity toward clupeine and its
esteratic activity with benzoyl-L-tyrosine ethyl ester (88).Since this in-
activation was reversed by 1.OM hydroxylamine and resulted in no loss
of homocitrulline residues, it appeared that the active serine residue had
reacted with potassium cyanate to inhibit the enzyme reversibly. A simi-
lar reaction had previously been observed with a-chymotrypsin (la?‘).
The a-amino group of subtilisin Novo also reacts with cyanate but has
no effect on activity since this residue remains carbamylated after re-
activation with hydroxylamine.
Although both the E - and a-amino groups of subtilisin Novo are free
to react with cyanate, their carbamylation does not disrupt the native
conformation of the molecule as judged by optical rotation and ultra-
126a. M. Ottesen, J. T. Johansen, and I. Svendeen, in “Structure-Function Re-
lationships of Proteolytic Enzyme,” (P. Desnuelle, H. Neurath, and M. Otteeen,
eds.), p. 175. New York, 1970.
127. D.C. Shaw, U‘.H. Stein, and S. Moore, JBC 239, PC67’1 (1964).
598 F. S . MARKLAND, JR., AND E. L. SMITH
centrifugation or alter the stability as a function of pH. Similar results

were obtained with subtilisin Carlsberg (101) ; carbamylation converted
7-8 lysines into homocitrulline residues and inactivated the enzyme. The
inactivation was reversed by hydroxylamine.
No change in specificity on the oxidized B chain of insulin was ob-
served with the succinylated or the carbamylated subtilisin Carlsberg.
With subtilisin NOVO,however, the initial rate of insulin hydrolysis was
decreased several fold for both the succinylated and carbamylated de-
rivative. The specificity was only changed slightly with the succinylated
derivative and was identical to that of native subtilisin Novo for the
carbamylated derivative.
B. METHIONINE
MODIFICATION
Treatment of a 1% solution of subtilisin Carlsberg a t pH 8.8 with

0.1M H20zresulted in the oxidation of a single methionine residue to
methionine sulfoxide (128) ; amino acid analysis indicated no other
change in composition. From a combination of tryptic and cyanogen
bromide digestions of the oxidized protein the modified residue was iden-
tified as Met 222, adjacent to the reactive Ser 221.
Oxidation of the specific methionine residue caused a loss of 91% of
the activity with acetyl-L-tyrosine ethyl ester; furthermore, there was a
linear relation between the degree of methionine oxidation and loss of
activity. I n comparing the kinetic parameters, it was found that oxida-
tion increases the K,n slightly for acetyl-L-tyrosine ethyl ester, benzoyl-L-
arginine ethyl ester, and cinnamoylimidazole while there is a slight de-
crease in K , with Cbz-glycine p-nitrophenyl ester. For all of these
substrates there is a decrease in kcat which is more pronounced for the
substrates with bulky side chains. Although the exact mechanism of the
changes in the enzymic activity brought about by oxidation of Met 222
is unknown, there are three possibilities: (1) alteration in conformation
of the enzyme, (2) alteration of the electronic environment around the
active site caused by changing from the hydrophobic thioether to a hy-
drophilic sulfoxide, and (3) steric effects. The last possibility appears to
be of little importance, a t least insofar as forination of the Gs complex
is concerned, since there are only slight changes in Km(app, between the
native and oxidized enzyme with various substrates. From the X-ray
crystallographic data it appears that the side chain of this methionine
residue swings away, about 1 A from the phenylmethane sulfonyl group
128. C. E.Stauffer and D. Etson. JBC 244,5333 (1969).

16. SUBTILISINS 599
when subtilisin B P N is sulfonylated (47). This indicates that the residue

does have freedom of movexnent and further excludes loss of activity by
oxidation on strictly steric irounds.
Earlier studies utilizing photooxidation had suggested that one methi-
onine as well as one histidine and one tryptophan residue were readily
oxidized causing rapid loss of enzymic activity (83). However, from
carboxymethylation of the oxidized derivative it was concluded that the
residue photooxidized was not the methionine residue next to the reactive
serine.
Ohtsuki et al. ( l d 8 a ) have shown that N-bromosuccinimide inactivated
subtilisin B P N presumably by oxidation of single tyrosine and, methio-
nine residues, although tryptophan was also modified.
C. TYROSINE
MODIFICATION
The finding by Glazer (39) that aromatic compounds act as competi-

tive inhibitors of the hydrolysis of aromatic ester substrates by the sub-
tilisins suggested the presence of a hydrophobic binding site in the
subtilisins. Additionally, the finding that the subtilisins exhibit higher
activity toward clupeine, but not small positively charged substrates
after treatment by reagents that alter the pK values of tyrosine phenolic
groups, suggested that certain tyrosine residues are involved in secondary
binding sites (129, 130).
Svendsen (130) reported that both nitration and iodination of sub-
tilisins Carlsberg and Novo increased the apparent activity toward
clupeine (for subtilisin Carlsberg a similar effect was also observed with
gelatin) , Also, succinylation of subtilisin Carlsberg increased the activity
with clupeine 6- to 7-fold. The increased activity probably results from
improved substrate binding since the K,,, for clupeine binding decreases
after nitration of either subtilisin Novo or Carlsberg. Both nitration and
iodination decrease the pK of the phenolic groups. The decreased pK is
probably related to the enhanced activity on the basic'protein, clupeine,
and suggests that one or more ionized tyrosine residues are at or near the
site of formation of the enzyme-substrate complex between clupeine
and subtilisin (126~) .
The activation of subtilisin Csrlsberg by succinylation is reversed by
treatment with hydroxylamine, and present evidence indicates that 14-
128a. K. Ohtsuki, C. L. Liu, and H. Hatano, J . Biochem. (Tokgo) 66, 863 (1969).
129. J. T. Johansen, M. Ottesen, and I. Svendsen, BBA 139, 211 (1967).
130. I. Svendsen, CompL. Rend. Trau. Lab. Carlaberg 36, 347 (1968).
16 threonine and serine residues have been succinylated. It may be sig-

nificant that in five instances a serine or threonine residue is located
next to a tyrosine residue in the sequence of subtilisin Carlsberg (Fig. 1) ,
offering the possibility of introducing a negative charge in the same spa-
tial location either by succinylation or (by lowering the pK of tyrosyl
residues) by nitration or iodination (130).
There is no promotion of activity, however, with tosyl-L-arginine
methylester since measurements of Km(app) and V,,,,, for this substrate
indicated only slight changes with derivatives of both subtilisins Novo
and Carlsberg. Furthermore, reduction of the nitrotyrosyl residues of
subtilisin Carlsberg to aminotyrosyl residues with sodium dithionite
caused the enhanced activity on clupeine to revert to that of the unmodi-
fied protein. This can be understood since the pK of the phenolic group
of aminotyrosine is about 10 (about the same as unmodified tyrosine)
whereas that of nitrotyrosine is about 7.
Analysis indicated that 6 of the 13 tyrosyl residues in subtilisin Carls-
berg had been modified after l-hr reaction with a 60-fold molar excess
of tetranitromethane at pH 8, with a maximum of 9 residues being modi-
fied after 18-hr exposure to a 120-fold excess of the nitrating agent. Even
after such long exposure, the enzyme maintained the 6- to 7-fold in-
creased activity and no evidence was obtained for modification of any
other amino acids, although tryptophan was not examined (130). With
subtilisin Novo only 3-4 of the 10 tyrosyl residues were nitrated under
the standard conditions. After exhaustive nitration about 8 of the tyro-
sine residues were nitrated without loss of activity. Iodination produced
essentially the same results with about 6 or 7 tyrosine residues in sub-
tilisin Carlsberg iodinated when maximal activation was obtained.
Subtilisin NOVO,however, behaved somewhat differently on iodination.
Four tyrosine residues could be iodinated readily with maximal activa-
tion toward clupeine, but when more iodine was added the enzyme was
rapidly inactivated. A total of 8 of the 10 tyrosine residues could be
iodinated (180).
Further characterization of the tyrosine residues in subtilisins was
obtained by spectrophotometric titration of the DIP derivatives (131).
The 10 tyrosine residues in subtilisin BPN’ and the 13 in Carlsberg fall
into three groups: 5 or 6 in both enzymes titrate normally with pKapp
of 9.7-9.9, 3 or 4 in both titrate with pK,,, of 11.25-11.6, and 2 or 3 in
both appear almost completely inaccessible to the solvent with pKapp
greater than 12.5. In 5 M guanidine hydrochloride all the tyrosine resi-
dues titrate normally with a pK.,, of 9.97 in both proteins. Markland
131. F.S.Markland, JBC 244,694 (19e9).

16. SUBTILISINS 601
(24, 132) has shown that 5 tyrosine residues in subtilisin Novo react
readily with tetranitromethane and an additional 3 or 4 react a t least
partially. Isolation of the peptides containing nitrotyrosine from com-
bined tryptic and cyanogen bromide digests has allowed precise identi-
fication of the nitrated residues (Table X ) . Tyrosine residues 6,21,104,
217, and 262 are fully nitrated, whereas residues 91 and 263 are partially
nitrated (see Fig. 1 ) . The tryptic peptide containing tyrosine residues
167 and 171 contains nitrotyrosine; however, it is not clear as yet
whether both residues are partially nitrated or only one is nitrated fully.
Presumably the 5 readily nitrated residues are the same as those that
titrate normally and those that are partially nitrated may be the resi-
dues with slightly higher pK,,,.
Nitration of subtilisin Novo with a fivefold molar excess of tetra-
nitromethane yields only one major nitrated residue which was identi-
fied as Tyr 104 (2.4).Nitration of this single residue did not fully pro-
mote activity on clupeine indicating that either a combination of several
nitrated tyrosine residues is necessary for full increase of activity or that
the nitration of a single tyrosine residue controls the activation but that
it is not the first to be nitrated. Weber and Kraut (133) have reported
that the first residue iodinated in subtilisin BPN’ is also T y r 104. Other
TABLE X
REACTIVITY
OF TYROSINE I N SUBTILISIN
RESIDUES BPN’ (OR Novo)
Residue No: Nitrationb Iodinationc
6 Full
21 Full Monoiodo
91 Partial
104d Full Diiodo
167 None or ? One is monoiodo
171 None or ?
2 14 None
217 Full Diiodo
262 Full
263 Partial
Of the 10 tyrosine residues, 5 titrate normally with pK.,, = 9.74, 3 with pK.,, =
11.25, and 2 with pK.,, > 12.5.
b Data from Markland (S4, 13%’).
c Data from Wright et al. ( 4 7 ) .
d This is the first residue to be iodinated or nitrated (94,f33).
132. F. S. Markland, Federation Proc. 28,877 (1969).

133. B. H. Weber and J. Kraut, BBRC 33,280 (1968).
iodinated residues appear to be 217, 167 or 171, and 21 as determined

by X-ray diffraction ( 4 7 ) (Table X ) .
Myers and Glazer ( 8 7 ) have ohserved that ili-acetylimidazole can
acetylate almost all of the tyrosinc residue iii the phcnylmethane sul-
fonyl derivatives of both subtilisins Novo and Cnrlsherg. Thus, the
tyrosines show no distinction in reactivity with this reagent in contrast
to the situation with spcctrophotomctric titration or nitration. It is con-
ceivable that the tyrosines that titrate in the first two groups (the nor-
mally titrating and those with slightly high pK,,,), and the two classes
of tyrosine reactive with tetranitronietliane (readily nitrated and only
partially nitrated) are all equivalent with respect to Ar-acetylimidazole.
I n subtilisin Novo this would comprise 8-9 residues leaving a t most 1 or
2 buried tyrosine residues. Solvent perturbation studies with 20% ethyl-
ene glycol indicated that 8.8 tyrosine and 1.6 tryptophan residues in
subtilisin Novo and 8.8 tyrosinc :tiid 0.8 tryptophan residues in subtilisin
Carlsberg wcrc exposed ( 8 7 ) .X-ray crystallography indicates that all
the tyrosine residues are on the surface of subtilisiri BPN' as well as the
3 tryptophan residues (4'7). The rcasons for these differences are not
apparent although there may be subtle interactions involving tryptophan
and tyrosine which are easier to measure in solution than to visualize in
the crystal structure.
VIII. Inhibitom Dye Binding
Study of the binding of a number of dyes a t the active sites of several

proteinases and other enzymes has shown that these interactions are
highly specific (%la). This results in part from the severe limitations
imposed on the geometry of the binding site because of the rigid struc-
ture of the dyes. Specific binding is found for Biebrich Scarlet ([6- (2-
hydroxyl-l-naphthyl) azo] -3,4'-azodibcnzene sulfonitte) to the active site
of a-chymotrypsin (134) and for thioninc (3,6-diaminoplienothiazine) to
the active site of trypsin (135).Thus, despite considerable similarities in
the three-dimensional structurw of these enzymes, these protein-dye
interactions are sensitive to the differences present in the active site
regions. Proflavine (3,6-diaminoacridine) binds to both of these enzymes
(136, 137). None of the above dyes interacts with the subtilisins.
134. A . N. Glazer, JBC 242,4528 (1967).
135. A. N. Glazer, JBC 242, 3326 (1967).
136. S. A. Bernhard and H. Gutfrrund. Proc. Natl. Acnd. Scz. U . 5'. 53, 1238
(1965).
137. S. A. Bernhard and B. F. Lee, Abstr. 6th Intern. Congr. Biochem. New York,
1964 Vol. IV, p. 297.
16. SUBTILISINS 603
I n contrast, Glazer (138) has found that 4- (4’-aminophenylazo) phenyl-

arsonate binds specifically to the active site of the subtilisins and does
not bind to the pancreatic proteinases. Spectrophotometric titration in-
dicated that the dye was binding in a time-dependent manner a t a single
binding site and with a similar spectral shift for the different subtilisins.
Assays showed a time-dependent loss in activity which paralleled the
spectral shift induced by dye binding. Dye binding was independent of
substrate concentration (with benzoyl-L-arginine ethyl ester or acetyl-L-
tyrosine ethyl ester). Indole (0.004M ) , a competitive inhibitor of the
subtilisins, did not inhibit the dye-protein interaction suggesting that the
initial binding site was distinct from the site involved in binding sub-
strates or competitive inhibitors. However, the dye was displaced by
phenylmethane sulfonyl fluoride indicating that the binding site was
spatially close to the active site of the subtilisins.
On the basis of the stringent stereochemical requirements of the pro-
tein-dye interaction, it can be concluded that the conformation of the
active site region of subtilisins NOVO,Carlsberg and Amylosacchariticus
is similar and largely unaffected by the differences in amino acid se-
quences. However, there are differences in the active site regions since
the K,, and V,,,,, values for a variety of ester substrates do show both
absolute and relative differences (Table VII) . Furthermore, the arsonic
acid dye failed to interact with trypsin and chymotrypsin, just as
thionine, proflavine, and Biebrich Scarlet failed to bind to the subtilisins.
Thus, the bacterial and pancreatic proteinases must possess different
surface characteristics in the general area of the catalytic sites of these
two groups of enzymes.
Certain features of the interaction of the phenylarsonate dyes with the
subtilisins appear noteworthy. Generally, the interaction of proteins with
small molecules is complete within seconds; the interaction of the sub-
tilisins with the arsonates is very slow, however, requiring from 15 to 60
min for completion depending on the concentrations of the reactants
(138).This suggested that the interaction involved either a slow confor-
mational change within a weak protein-dye complex that was initially
formed very rapidly, or a slow direct interaction of the bound dye with a
group involved in catalysis. The protein-dye interaction could be re-
versed by dilution or by suitable change in p H (138).
Studies with simpler phenylarsonate analogs of the dye, such as
phenylarsonate and its p-nitro, p-amino, and p-methyl derivatives,
showed that these compounds were also time-dependent inhibitors of the
subtilisins with an initial site of hinding distinct from the substrate l h l -
ing sitr. Unlike the large phenylar~onatcdye, the smaller phenylarso-
138. A. N. Glazer. Proc. Natl. Acnd. Sci. U.S. 50, 996 (1968).
nates were effective inhibitors of a-chymotrypsin and some of them of

trypsin also (139).
The pH dependence for inhibition of the subtilisin Novo catalyzed hy-
drolysis of N-benzoyl-L-srginine ethyl ester by the phenylarsonate dye
followed the theoretical curve for the protonation of a group with a pK:
of 7.17. With the simpler phenylarsonates as with the dye, the presence
of substrate, even a t a 100-fold molar excess over the inhibitor, is with-
out effect on the rate and extent of inhibition. This suggests that all of
the arsonates bind at a site distinct from the substrate binding site form-
ing an initial complex that is still catalytically active and that this com-
plex then undergoes a slow rearrangement to give an inactive enzyme as
shown by Q. (4) :
E I +
fast
(E1)acti”e -
slow
(E1’)inactive (4)
From the pH dependence of inhibition of the subtilisins by the phenyl-
arsonates it would appear that the binding of the inhibitor requires the
presence of a protonated histidine residue at the active site. A possible
scheme for the interaction of the phenylarsonates with the subtilisins is
presented in Fig. 7. The initial reaction (Step 1) is envisaged as depend-
ing on an electrostatic interaction between the phenylarsonate anion and
a protonated histidine residue, as well as a hydrophobic interaction in-
volving the aromatic ring. Subsequently (Step 21, ester formation may
take place with the active site serine residue. This mechanism is con-
Step 1
CHI-OH
-NH-CH-CO-
I
Q HC=C-CH,-CH
I I
I
NH
I
I
co
H O / t b H+Nbc/NH
H I
Step 2
HN,
+ ‘6 ,NH CO
I
-NH-CH-CO-
FIO.7. Possible mechaniam for the formation of an arsonic acid ester with the
reactive serine in the subtilisins. From Glazer (199).
139. A. N. Glazer, JBC 243, 3693 (1968).

16. SUBTILISINS 605
sistent with all the available data but awaits definitive proof from
X-ray crystallography.
IX. Other Beci//ur Alkaline Proteinaser
As already noted (Section 11), Keay and Moser (IS) were able to
group the highly purified subtilisins into two classes on the basis of
amino acid composition, serological cross-reactions, and the ratio of
esterase to proteinase activity. It remains to be determined whether
other alkaline proteinases will fit this classification.
There have been some reports describing the partial characterization
of alkaline proteinases from a variety of different strains of Bacillus.
Among these are B . cereus, strain K, 931 which produces three alkaline
proteinases ( 1 4 ) . Bacillus licheniformis culture filtrates also produce
three proteinases, one of which is certainly of the subtilisin type since
it had high esterase activity with N-acetyl-L-tyrosine ethyl ester and
much lower activity with N-benaoyl-L-arginine ethyl ester, exhibited an
alkaline pH optimum, and was inhibited by both DFP and phenyl-
methane sulfonyl fluoride (141).
A transforniable strain of B. subtilis (strain 168 indole-) produced two
different proteinases (designated basic and acidic based on their binding
to DEAE-cellulose) at different times in the growth cycle (14.9). Both
were inhibited by DFP and phenylmethane sulfonyl fluoride, although
the basic one did not possess appreciable activity on benzoyl-L-tyrosine
ethyl ester. Amino acid analysis showed significant differences between
the two proteinases although the basic proteinase had a composition
that was not unlike that of subtilisin Carlsberg. More recently it was
shown that the basic proteinase appeared similar kinetically and im-
munologically t o subtilisin Nova as well as having some antigenic
regions in common with subtilisin Carlsberg whereas the acidic pro-
teinase appeared altogether different from the other subtilisins (143).
Under conditions for production of the acidic proteinase a high molecular
weight proteinase was also present; this enzyme waa devoid of esterase
activity on benzoyl-L-tyrosine ethyl ester. Both the acidic and basic
proteinases were acid labile and had similar p H optima around pH 7.5-
140. Y. Furukawa, Y. Fujii, and H. Takahashi, Agr. Biol. Chem. (Tokyo) 32, 832
(1968).
141. F. F. Hall, H. 0.Kunkel, and J. M. Preacott, ABB 114, 146 (1960).
142. H. W. Boyer and B. C. Carlton, ABB 128,442 (1988).
143. J. H. Hageman and B. C. Carlton, ABB 139,67 (1970).
8.0. The significance of the production at different times in the growth

cycle of the two proteinases is not clear.
Rappaport e t al. (144) isolated a DFP-sensitive proteinase from the
same strain of B. subtilis as Boyer and Carlton (142); however, its
amino acid composition was different from either of those described later
by Boyer and Carlton. This proteinase in its native form has a sedi-
mentation coefficient of 9.35 S, but a t pH 2.3 the aggregated material
broke down into 1.8 S inactive subunits consonant with a molecular
weight of 28,800 calculated from the amino acid composition. Alanine
was obtained as the NH2-terminal residue although only in 30% yield.
Serine and leucine were liberated most rapidly by carboxypeptidase A
and reached levels of 0.92 and 0.9 mole per mole of protein. Riggsby
and Rappaport (146) reported that the active proteinase had a molecular
weight of 1.66 x lo6 and that either very alkaline or very acidic con-
ditions converted the active species into inactive units of molecular
weight 27,600. Some evidence was presented for the occurrence of a
14,000 molecular weight subunit ; however, the possibility of autolysis
or digestion by a contaminating proteinase cannot be excluded.
Ganno (146) has reported a proteinase from another strain of B .
subtilis. This proteinase is optimally active at pH 7.5-8.5 and is not
inhibited by EDTA or common thiol reagents (the effects of DFP were
not reported). The molecular weight and amino acid composition are
similar to those of the subtilisins but the amino acid analysis indicated
only 244 residues. One interesting difference between this enzyme and
the subtilisins is the extremely low isoelectric point a t pH 3.
Millet ( 1 4 6 ~ characterized
) three proteolytic enzymes from the Mar-
burg strain of B. subti2is during sporulation. One of these enzymes ap-
pears similar to the subtilisins in that it is inhibited by DFP, shows
optimum pH activity in the neutral to alkaline range, shows esterolytic
activity on p-tosyl-L-arginine methyl ester and benzoyl-L-tyrosine ethyl
ester, and is not inhibited by EDTA.
X. Practical Uses of Subtilidns
In general the applications of the subtilisins have been mainly twofold:

for determination of the primary structure of other proteins, and more
144. H. P. Rappaport, W. S. Riggsby, and D. A. Holden, JBC 240, 78 (1965).

145. W. S. Riggsby and H. P. Rappaport. JBC 240,87 (1965).
146. S. Ganno, J . Bwchem. (Tokyo)58, 556 (1965).
146a. J. Millet, J . Appl. Bacterial. 33, 207 (1970).
16. SUBTILISINS 607
recently, as an additive to laundry detergents to improve their cleaning

power by hydrolysis of fabric bound proteins.
A. PROTEIN
SEQUENCE
Probably the first practical use for subtilisin was in the determination
of the amino acid sequence of oxytocin ( 9 9 ) .Subtilisins have also been
used for the hydrolysis of a variety of other proteins and peptides.
Among these were the limited digestions, already mentioned, of oval-
bumin (147) and ribonuclease (104).Other uses may be noted briefly:
the digestion of native hemoglobin ( l o ) , the determination of the
amino-terminal sequence of a-crystallin (I@), the identification of
citrulline in the medulla protein of the quill of the African porcupine
(150),the determination of the sequence of the nonapeptide phyllocae-
rulein from the skin of a South American amphibian (161), as an aid
in distinguishing the differences in sequence between beef and pig insulins
( 9 8 ) ,and as an aid in determining the positions of the disulfide bridges
linking the two chains of insulin ( 9 7 ) .Foster and co-workers (156, 153)
have used subtilisin BPN’ to probe the structure of bovine plasma
albumin by limited digestion of an albumin-sodium dodecyl sulfate
complex. Hill (154) has presented an extensive discussion of the hydrol-
ysis of a variety of proteins by the subtilisins, and the articles by Smyth
(155) described the procedures to be employed in using subtilisin for
sequence work.
Because of their broad specificity, the subtilisins have been useful
for studying the sequences of small peptides which are not hydrolyzed
by more highly specific proteinases.
A water-insoluble active derivative of subtilisin Novo has been pre-
pared by cross-linking with glutaraldehyde. Amino acid analysis in-
dicates that lysine residues are probably involved in the intermolecular
cross-linking reaction with glutaraldehyde. This preparation, when mixed
with cellulose powder in a chromatographic column, has shown some
promise in digesting proteins (166).
147. M. Ottesen, ABB 85, 70 (1956).

148. M. Ottesen and W. A. Schroeder, Acta Chon. Scand. 15, 926 (1961).
149. H. J. Hoenders, J. Van Tol, and H. Bloemendal, BBA 160, 283 (1968).
150. P. M. Steinert, H. W. J. Harding, and G. E. Rogers, BBA 175, 1 (1969).
151. A. Anastasia, Expenentiu 25, 8 (1969).
152. B. J. Adkins and J. F. Foster, Biochemistry 4, 034 (1965); 5, 2579 (1968).
153. D. M. Pederson and J. F. Foster, Biochemistry 8,2357 (1969).
154. R. L. Hill, Advan. Prolein Chem. 20, 37 (1965).
155. D. G. Smyth, “Methods in Enzymology,” Vol. 11, pp. 214 and 421, 1967.
156. K. Ogata, M. Ottesen, and I. Svendsen, BBA 159, 403 (1968).
B. USE IN DETERGENTS
The alkaline pH optima, stability to anionic detergents and to rela-

tively high temperatures have made possible the use of the subtilisins
as additives to household anionic detergent products. Thus, the housewife
has become an amateur enzymologist and has been able to see a demon-
strable benefit in the removal of certain formerly stubborn stains and
soils. Laboratory tests and practical experience have indicated that the
proteolytic enzymes alone do not give a satisfactory cleaning perform-
ance; protein spots are not satisfactorily removed by the action of
the enzymes alone. Rather, they must be used in combination with
detergents and in so doing a marked synergistic cleaning action is
obtained, far superior to that resulting from summing the individual
cleaning actions of the enzyme and the detergent alone.
Although figures are not readily available it appears that the alkaline
proteinases, known collectively as subtilisins, are being produced in far
greater quantities for commercial use than all other enzymes together.
I n view of the increasing practical use of subtilisins and related enzymes,
we may expect a great surge in the study of new enzymes of these types
in the future.

Markland 1971

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Subtilisins: Primary Structure.

Chemical and Physical Properties

X. Practical Uses of Subtilisins . . . . . . . . . go6

II. Historical Background and Development

The extracellular alkalinc proteinase, subtilisin, was discovered by

1. B. Hagihara, “The Enzymes,” 2nd ed.. Vol. 4, p. 193, 1960.

In 1960, Ottesen and Spector (9) reported on the characteristics of yet

111. Physical, Chemical, and Stability Properties of the Subtilisins

Guntelberg and Ottesen (7) reported that subtilisin Carlsberg had an

E2nm 11.7 8.6 11.7 11.9

From the amino acid sequence.

physicochemical properties of subtilisin Carlaberg. The amino acid

Subtilisin BPN’ was shown to have essentially the same stability

19. F. S. Mark1:ind and E. L. Smith, JBC 242, 5198 (1967).

Subtilisin Novo was shown to be similar in physicochemical proper-

Tsuru and his co-workers (10 reported that subtilisin Amylosac-

(identical to that of subtilisin BPN’), and the amino acid composition

IV. Primary Structure of the Subtilirinr

Although many of the physical properties of the subtilisins are similar

28. J. A. Hunt and M. Ottesen, BBA 48,411 (1961).

38. F.Sanger and D. C. Shaw, Nature 187,872 (1960).

Tyr (one Tyr-Tyr sequence). Additionally, there are many Ser-Thr or

The chromatographic behavior and compositions of the 14 expected

The complete sequence of subtilisin Carlsberg (@) was determined

The partial sequence of subtilisin Amylosacchariticus (24) is shown

Gly. Asn.Ser.His*.Gly. Thr.His, Ile. hla. Gly.Thr.Val,Ala. Ma. Leu, .~n.Asn.Ser.Ile.Glg.Val.Leu,Gly.Ser,hla,Pro.Ser,Ser,Ala,Le~~,

Tyr)(Ala.Val) Lys ~Yal.I~u.Thr,Asp,Ile.Gly.Ser.Gly.Gln.Tyr.Ser.Trp.Ile)(Ile,~n.Gly.Ile,Glu)Trp(.\la,Ile,Ser..\sn,Asn)~let

There are only 4 methionine and 12 basic residues in subtilisin Amylo-

In comparing the sequences of subtilisins B P N and Carlsberg and the

Chymotrypsin A (bovine) hr-Leu-Leu-Lys

tional events in the nucleotide codons in this region. Nevertheless, the

Changes Subtilisin Carlsberg changed

Lysine 11 36 Asn (2),Thr (l),TYR (1)

servative in nature. It is evident from the information in Table I V that

repetitions; e.g., Ala-Ala-Ala (residues 151-153, 230 through 232, and

V. Active Site Studies

The involvement of serine in the active site of subtilisins Carlsberg

51. H. Matsubara. R. M. Sasaki. and R. K. Chain, Proc. Natl. Acad. Sci. U. s.

activated by DFP. Tsuru and co-workers also showed that DFP is a

1. Serine Sequence in Other Proteinases

2. Conversion of Active Serine t o Cysteine

Various mammalian enzymes Asp-Ser'-Gl y

63. D. C. Shaw, Ph. D. Dissertation, University of Cambridge (1962).

Koshland ( 7 1 ) . In both cases the proteinase was reacted first with

ited by p-mercuribenzoate, whereas this hydrolysis catalyzed by native

The involvement of histidine in the activity of proteolytic enzymes

81. E. A. Barnard and W. D. Stein, Advan. Enzymol. 20,51 (1958).

with subtilisin B P N with a concomitant loss of activity. The reaction

Chymotrypsins A and B (bovine) Val-Thr-Ma-Ma-His'-Cy s G ly-Val-Thr

92. L. B. Smillie and B. S. Hartley, BJ 101, 232 (1966).

several mammalian proteinases) (46,92). This single amino acid replace-

FIG. 5. Comparison of the reactivity of subtilisins Carlsberg and Amylosac-

92a. K. Morihara and T. Oka, ABB 138,526 (1970).

inactivation was partly prevented by the presence of hydrocinnamic acid.

VI. Substrate Specificity and Enzymic Properties

Tuppy ( 9 6 ) ,Haugaard and Haugaard (97), and Meedom (98) studied

differently from subtilisin Carlsberg. Nonetheless, all three proteinases

The subtilisins show low specificity when tested on a wide variety of

whereas the last two were kinetically indistinguishable. By comparison

Amylosacchariticu9 Novae Carlsberg BPNtd

f No dioxane in reaction mixture.

Inhibitor Carlsberg Novo BPN’