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Papain
Papain
Papain
Enzyme chemistry is an integral part of any introductory biochemistry course and is an important aspect of most
undergraduate protein chemistry courses. By studying the
action of enzymes, students learn to appreciate the dynamic
nature of proteins as well as their role catalyzing myriad
chemical reactions in biological systems. Much time in class,
especially at an introductory level, is spent investigating the
catalytic cycle and the role of the various amino acid residues in the active site of the enzyme. While there are numerous enzyme systems that can be studied, proteases are
often used in class as well as in teaching laboratories because
they are relatively simple, inexpensive, and well understood.
In spite of this, very few undergraduate experiments exist that
directly explore the chemistry of the amino acid residues in
an enzymes active site. (These readers found none.)
Many biochemical techniques such as affinity labeling,
radioactive tagging, and fluorescent and spin labeling take
advantage of the chemical specificity of residue side chains
to probe protein chemistry phenomena (1). Affinity labels
are used to identify active site residues important in catalysis, and spin labeling is employed as a general method for
exploring the conformation of membrane and soluble proteins (2). The reactive side chains of aspartate, glutamate,
lysine, serine, threonine, tyrosine, histidine, and cysteine can
be targeted with a wide variety of reagents.
The experiment we are proposing uses methyl
methanethiosulfonate (MMTS) and phenylmethylsulfonyl
fluoride (PMSF) to specifically modify the cysteine and serine
residues in the active sites of papain and subtilisin, respectively (35). Both enzymes are subjected to the same series
of treatments, and the differences are noted. The chemical
modification of papain by MMTS causes a complete loss of
activity, and when the active site cysteine is regenerated, the
return of activity is noted. Since subtilisin is a serine protease, treatment with MMTS has no effect on it. However,
treatment of subtilisin with PMSF results in an irreversible
loss of activity while the sulfonyl fluoride does not dramatically affect papain under the reaction conditions used in this
experiment.
The comparison of papain and subtilisin accomplishes
many desirable teaching goals. The active site differences between the two proteases are displayed. This opens many possibilities for discussion regarding the reactivity of cysteine
versus serine and the role of cysteine in proteins. (Subtilisin
contains no cysteine while papain has seven.) The covalent
modification of the enzymes and subsequent rescue of papain shows the beginning biochemist that proteins can be
manipulated structurally and that this directly affects their
functioning. In addition, students will appreciate the ease of
manipulating the two enzymes as if they were simple chemi1048
papain
and
NH3
papain
about 90%
SH
about 10%
excess
cysteine
COO
papain
SH
H3N
S
S
COO
almost 100%
NH
cystine
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In the Laboratory
nitroanilide while papain was assayed using N-benzoyl-DLarginine p-nitroanilide hydrochloride. MMTS was diluted in
acetonitrile (CH3CN), and PMSF was used as a stock solution in ethanol. All modification reactions were conducted
in acetate buffer at pH 5.0. Dialysis was done in 5 mM acetate buffer at pH 5.0 with 50 mM NaCl using a molecular
weight cutoff of 6,000 to 8,000.
All enzyme solutions and modification reactions were
prepared and conducted in polypropylene tubes fitted with
snap-on polyethylene caps. All enzyme assays were done in
disposable polystyrene cuvettes. The substrate stock solutions
as well as the dilution of MMTS and PMSF were made in
siliconized micro centrifuge tubes. A detailed experimental
procedure is available in the Supplemental Material.W
Hazards
Protein powders are allergenic so students were given
polypropylene tubes with 20 mg of enzyme. They could then
add the acetate buffer directly to the tube at the beginning
of the experiment. CH3CN can irritate eyes, nose, throat,
and skin. DMSO is absorbed directly through the skin and
acts as a solvent carrier for the transport of any substance in
solution. MMTS has a strong odor and is irritating to the
eyes, skin, and respiratory system. In addition, PMSF burns
the skin and eyes, and is very destructive to mucous membranes. Gloves, labcoat, and eye protection should be worn
when manipulating these four chemicals. The solution of
MMTS in CH3CN should be manipulated in a fume hood.
Results and Discussion
Commercial papain often contains a significant quantity of the mixed disulfide of papain and cysteine (6). As a
papain
SH
MMTS
MMTS
PMSF
O
S
papain
CH3
O
subtilisin
O
100 % yield
unchanged
and
O
S
OH
subtilisin
100 %
PMSF
O
S
papain
OH
subtilisin
unmodified papain
O
MMTS H3C
CH3
PMSF
CH2
Scheme II. Treatment of papain and subtilisin with MMTS and PMSF.
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1049
In the Laboratory
Table 1. Student Activity Resultsa for the Reaction between the Substrate and the Enzyme as Measured by the
Absorbance of p-Nitroaniline
Subtilisin and
N-Succinyl-L-Phe p-nitroanilide
Abs
Papain and
N-Benzoyl-DL-Arg p-nitroanilide
hydrochloride
Abs
Commercial enzyme
0.382
0.294
0.354
2.134
Enzyme Condition
MMTS
PMSFb
MMTS
PMSFb
0.312
0.007
0.016
1.35
0.294
0.006
2.09
2.77
The activity is measured by the magnitude of the p-nitroaniline absorbance at 410 nm. Data are the average from five students results.
The comparison with PMSF has not yet been attempted with students. The authors obtained these results.
1050
Supplemental Material
Detailed instructions for students and notes for instructors are available in this issue of JCE Online.
Literature Cited
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2. Columbus, L.; Hubbell, W. L. TiBS, 2002, 27, 288295.
3. Singh, R.; Blttler, W. A.; Collinson, A. R. Methods Enzymol.
1995, 251, 229236.
4. Gold, A. M.; Fahrney, D. Biochemistry 1964, 3, 783791.
5. Whitaker, J. R.; Perez-Villasenor, J. Arch. Biochem. Biophys.
1968, 124, 7078.
6. Klein, I. B.; Kirsch, J. F. Biochem. Biophys. Res. Commun. 1969,
34, 575581.
7. Singh, R.; Blttler, W. A.; Collinson, A. R. Anal. Biochem.
1993, 213, 4956.
8. Roberts, D. D.; Lewis, S. D.; Ballou, D. P.; Olson, S. T.; Shafer,
J. A. Biochemistry 1986, 25, 55955601.
9. Hsia, C. Y.; Ganshaw, G.; Paech, C.; Murray, C. J. Anal.
Biochem. 1996, 242, 221227.
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