TEE JO~RXAL 01’ BIOLOGICAL CHFIMISTRY

Vol. 244, SO. 16, Iswe of August 25, pp~ 43754381, 1969
Prinkd in C.S.A.

Porcine Heart Lactate Dehydrogenase

OPTICAL ROTATORY DISPERSIOS, THERhIODl-XAMICS, AND KINETICS OF
BISDISG REdCTIONS*

(Received for publication, April 11, 1969)

SUMMARY Zion (T), and chemical relasation (8) points to difkences in

The tinetics of NADH, 3-thionicotinamide adenine conformation between apoensyme, binary complex n ith S-IDE-I,
dinucleotide (TNAD), and oxamate binding to the H4 and ternary complex with osalatc. In ihe H, i;oenz>-me, how-
isoenzyme of lactate dehydrogenase from the pig has been cvcr, t.hc information is less condusive.
investigated by temperature jump techniques. The dis- Halhavap and Criddle (9) have shown with borine H, lactate
sociation rate constant for TNAD is considerably larger than dehydrogenase that., at. low enzyme concentration;, ~Jy.Kate

for NADH, whereas the recombination rate constant is c:.~ affect. the quarternary structure of lactate deh>-drogennsc.
smaller for the oxidized coenzyme than for the reduced Stopped flon- experiments with the 84 isoenzpe of Ijig suggest

molecule. The kinetics of oxamate binding agrees satis- that a. structural change occurs either in the enzyme-S-1DH
factorily with a simple binding mechanism. The optica con~plcs or in the enzyme-NhI~H-l,gruvatc comples during
rotatory dispersion spectrum of the protein, which indicates catalysis (10). Di Sabato and Ottesen (11) ha\-e interpreted
a-helical content, is unperturbed by binding of NADH and t.heir experiments on hydrogen-dcut,erium eschange as eridence
for a structural change in the complex of S.%DH rrith the chicken
oxamate, or by changing the pH from 6 to 8. Both the
enthalpy and entropy of oxamate binding are strongly pH- llriirt II4 isoenzyme.
dependent. The interaction of this inhibitor with the protein In order to obtain further data regarding ttructural changes

cannot be explained simply in terms of an electrostatic in tl-tc Hq isoenzyme of lactate dehydrogena:e and 10 clarif!- ccr-
attraction+ Other effects, primarily entropic, are of large bait1 questions regarding its mechanism, the present inI--eAga-
importance. Evidence is presented which indicates that tious were undertaken.
TNAD in solution exists in two conformational forms which
are in rapid equilibrium.
_llnfeiia7s--Pig heart lactate dehydrogena?c m-as a product of
C. E’. Ho&ringer und Soehne, Mannheirn, II-est Germany. The
enzyme isolated from pig heart is knon-n to conrain all fire isocn-
zymes (la), but the proportion of H, isoenzymc i< very high
(over 90%) (13). That the Uoehringer enzyme does contain A
The i>ot~nx~~mes of l:lctute dehydrogenace (r~lnctatc:SAID Inrgc fraction of a single, rapidly migrating nnndic compolJent
osidoreduct:i5e, EC 1 . 1,1 .25) differ from one another in numcr-
at, $1 6.8 Tvas shown by T-ertical polpncrylamide gel electro-
ous w-n)-S (1). Some of the most prominent dissimilarities are
It has been established that this compontnt Ha
I)Iloresis. ia he
in ihoir catalytic mechnnimx. The results irom steady state
isoenz:-me (12). The appearance of the electrophoretoprams
kinelics indicate that slructwal change.5 pl;t>- a ejgnificant role
XV WV>- similar to that in a publiahcd report 011 pig heart Iac-
in cxtalyh hy the 11 :I irocnzyme (9) but not I.)!- the H: isocu-
t:ite dchydrogenase electrophoresis which ~-a-’ carried out in
zymc (4)~ In \-ieTv of offorts to determine the struclure of this
st:wch gel (14). The molecular weight tind other J)h!-sical prop-
enzyme b?d s-ray crystallography (j), it. is imporinllt to ascert&n
erties of the Hd isoenzyme of pig heart n-ere recentI>- determincd
by olhrr t!-pes of ex+hJlcC whether substr~lte-tlependcnlr con-
to a high degree of accuracy (15).
form;ltional changes occur in the various isocnzymes.
Recause a small proportion of isocnz:-me.5 other than the H4
In the ca’e of the AI4 isocnzyme, t.he result:: teem clear. El-i-
iso,nz?-me is present in the Bochringer preparation, it was neces-
dencc from fluorometric titrations (6), optirfll rolator~- cliqwr-
s::\~J- to determine n-he&r these other isoenz>-me: \\mould affect
* This investigation ~1-3s supported in pars b?- Publir Health the kinetic and equilibrium investigations. Tbc enzyme was
Service J?eIlol\ship G-F?-A>I-32, 6734192 (raj from the Sntionltl l>ruified by fractionation on a column of I)E.G%ephades (16).
Inslitute of Arthritis and Metabolic Diseases, and in part by a I<cy kinetic experiments were then checked n?th the purified Hd
fellowship from the Europcnn MoIeculsr Biology Organization.
: dddrex to n-hirh inquiries should be sent, component. Xo differences were found from the rc4ts ob-
l The 17~3 of the Icttcrs II and ill to denote tn-n tiitrerent types of tnincd n-ith unresolved lactate dehydrogenase.
subunit in Iactnte deh>-drogcnase m-as proposed b>- Cahn ef cri. (Z), For the preparation of enzyme stock solution:, the 5u%penaion

4375
This is an Open Access article under the CC BY license.
4376 Lactate Dehydrogenase Vol. 244, No. 16

of crystals was centrifuged at 30,000 X g, and the precipitate model 14 or an Eppettdorf rccordittg sl)cctrol)hotonleter equipped
was dissolved in the appropriate cold phosphate buffer. Washed, with thermostated cell holders. Measurements of pH were
activated charcoal (approxitnately 1 trig of charcoal per mg of m&e with a Radiotneter 22 pH meter. The high voltage clcc-
enzyme) was added to the solution,2 and the suspension was trophoresis apparatus was produced by the E-C Apparatus
swirled gently for 10 min at rootn temperature, then centrifuged. Corporation, Philadelphia, 1’etmsylvattia.
The supernatant solution, filtered through a Millipore filter, was Methods-The cottcentmtiott of active sites in enzyme stock
dialyzed for several hours against the desired buffer. The result- solutions was measured before each day’s runs by titration with
ing enzyme stock solutions had A 2m:-.1Zmratios of 1.98 to 2.04 and NA1l)+ and sulfite ncscordittg to the method of Holbrook (19).
a specific activity of approximately 360 mlits per mg, when Coenzyme and osamate stock solutions were freshly prepared
tested according to Biocllernica Informationen, I{oehringer. daily.
When kept cold, enzyme solutions were stable for a few days. For the determination of X, and b0 from spectropolarimrtric
lttorganic buffers and salts were reagent grade. NADH, data, the wave lengths were scanned from 600 to 350 nm and
NAD+, TNAD,3 and sodiutn ljyruvate were Boehringer product,s. 600 to 310 nrn, respectively; X, and b. were extracted from Yattg-
Sodium oxamate, obtained from .ildrich, was recrystallized Doty (20) and Moffitt-Yang (21) plots. 111 the b0 calculations,
from ethanol-water. XO was taken at 212 nm and the tneatt residue weight was calcu-
Activated animal charcoal was washed by boiling twice with lated to be 101 from a published atnitto acid analysis for the Hq
12% HCl, after which it was rinsed thoroughly with water, 2 11 isocttzyme of the pig (22).
KCX, and water, then dried. Dialysis tubing was washed ac- Tetnpcrature jumps were approxinlately 3%5”, depending on
cording to the method of Anderson and Weber (6). the initial temperatures of the solutions. A maximum of four
dpparatus-Fluorometric titrations were carried out, in a to six jumps was made with each solution, and the relaxation
thermostated Aminco-Bowman spcctrofluorometer. Additions titnes were calculated frotn plots of log (St - SW) versus time,
of NAl)H to enzyme test solutions, or of oxatnatc to enzyme- where St is the observed fluorescettce or transmitted intensity
NADH solutions, were made with an Agla microsyringe as- in millivolts at titne t. There was no detectable alteration of
sembly (Burroughs Wellcome). Spectrophototnrtric titrations enzyme activity after four to six temperature jutnps.
with TPIlTAD were done in a thermostated Zeiss I’MQ II spec- Fluorotnetric titrations were performed according to previousl)
trophotometer. Optical rotatory dispersion tneasurcments were described tnethods (23, 24).
performed in a Cary model 60 spectropolarimeter containing a
thertnostated cell holder. RESULTS

The kinetics of N;ZDH and oxamate binding to lactate dehy-
drogenase was followed in a temperature jump apparatus by
observing the changes in protein or NADH fluorescence following
the temperature perturbation. A high pressure Osram lamp,
either 100 watt mercury or 75 watt xenon, was used for excita-
tion. The desired excitation wave length was selected by means
of a Bausch and Lomb high intensity grating tnonochromator.
Fluorescence was observed at 90” with an RCA 11’28 photo-
multiplier. The temperature jutnp cell contained four quartz
windows. Concave mirrors were placed at locations facing the
directions of excitation and emission, so that the intensities of
exciting and emitted light were essentially doubled.
(Jenaer Glaswerk Schott und Genossen, Mainz, West Germany)
were used to select the desired emission bands. Except for these
optical modifications, the temperature jump apparatus was
constructed as previously described (18).
Temperature jump investigations of TNAD binding to lactate
dehydrogenase were performed by observing the changes in
TNAD absorption at 360 nm that were associated with enzyme
binding.
Enzyme activity determinations were made in either a Cary
2 Treatment with charcoal is required for accurate kinetic meas-
urements. If charcoal treatment was not performed, the
A280:A260 ratio was found to be approximately 1:7. Under these
conditions, temperature jump investigations gave rise to slow re-
laxation effects. From the concentration dependence of these ef-
fects and the essential constancy of the 280:260 ratio through
dialysis, it could be inferred that a nondialyzable impurity was
present in the commercial preparations, although the enzyme was
highly active under the test conditions. After the same enzyme
solution had been treated with charcoal, a high 280:260 ratio was
observed and the slow relaxation effects disappeared. The pos-
sible nature of this impurity has been discussed (17).
3 The abbreviation Itsed is: TNAD, 3-thionicotinamide adenine
dinucleotide.
Equilibrium Titrations-The spectrofluorotnetric titrations of
lactate dehydrogenase with NADH and of lactate dehydrogenase-
N.\DH with oxatnate yielded values for the dissociation constants
which were in good agreement with published values for other lac-
tate dehydrogenases (6, 23-27), indicating that bhe equilibrium
binding properties of pig Hq lactate dehydrogenase with these com-
pounds are similar to the binding properties of lactate dehy-
drogenase isolated from a variety of other m4mmalian tissues.
The equilibrium constants for TNAD binding agreed well with
published measurements of the NhD+ binding constants wi-ith
other lactate dehgdrogenases (26, 28). The results of the titra-
Filters tion experiments are compiled in Table I.
The thertnodyttamics of osamate binding, determined from
van’t Hoff plots (Fig. I), are listed in Table II. Throughout
the temperature range investigated, the effect of pH on t,he dis-
sociation constant,s is small. It is evident, however, particularI>
at lower temperatures, that the affinity of the enzytne for oxamate
is greater at pH 7 than at pH 6. These findings suggest that
the interaction of osamate with the protein is not solely an elec-
trostatic attraction. 111 contrast, the results of Winer and
Schwert (24) indicate that in bovine H4 lactate dehydrogenase
the binding of oxatnate is greatest at pH 6 and decreases at, all
higher pH values.
A1 further difference frotn the bovine Hb isoenzyme is in the
enthalpy of oxamate binding, which is pH-dependent. Winer
and Schwert (24) stated that in the bovine enzyme the value
of AH0 is unchanged in Tris buffers between pH 7.10 and 9.51.
The value of AH0 determined in the present experiments at
pH 7 is within experimental error identical with the value of
Wirier and Schwert for pH 6.80 in 0.1 M phosphate (24).
The absolute magnitudes of the oxamate binding constants in
phosphate buffer for the bovine (24) and pig enzytnes netlrly
coincide at pH 6, but, at higher p1-I the pig enzyme appears to
Issue of August 25, 1969 H. d’A. Heck 4377

hare :I greater affinity for this substrate analogue. Thus, despite TIULE III
several kinetic similarities (10, 29), bovine and pig lactate dehg- Oplical roln/o~‘!/ dispersion parnmetos of pig heart lactate rlehydro-
drogenase differ in several wags with respect to osamate binding. genase in 0.1 JI sodium phosphate, with ionic strength of 0.3 x
~Lpprosimate values for the cnthalpies of cocnzyme binding with AYaCI
were obtained from titration data and chemical relaxation esperi-

T.\T3LE I mn

.lIic~,omolar tlisrociation c,ons(anls for pig hcwt lactate dehydrogen- 6.0 18G f 113” 264 f la
me in 0.1 .II sotli~rr~ phosphale,
wilh ionic strength of 0.3 .+I wilh 6.5 186 + 10 258 zk 3
.Yn(‘l, from ,jirtorome(ricand sper/,opho/o),aetrir lilralions 7.0 214 f 38 25G 27 12
7.5 160 ZtZ3-l 247 z!z 6
PII I KNADH= 8.0 217 f 31 250 zt 6

17.8 f 2.(ir 1350 I-t 5OCl’” n Probable error.

6.00 1.2 z!z 0.3c
1135 f 500

8.00 0.56 zk 0.25 25.7 f 3.8

n 20’; excitation and emissioll wave lengths, 295 aud 430 nm,
respectively.
b At, 20”; exritat ioil and emission wave leiigths, 340 and 445
nm. respectively. The concentration of free NADH in these
experiments exceeded KNADA by 20.fold.
T
c Probable error. ”
d 20”; spectrophotometric titration at 360 nm. %
e ilt 3”.

I I I

2 4 6 8 IO

([-I
Sites free + [NADH])X to6

FIG. 2. Plots of the reciprocal relaxation times against concen-

trations for the system lactate dehydrogenase + NADH in 0.1 M
sodium phosphate, with ionic strength of 0.3 M with NaCl. Lines
were drawn by the method of least squares. 0, 20”, pH 6.00; 0,
20”, pH 8.00; A, 3”, pH 6.00.

3.30 3.40 3.50 3.60

800 -
103/T
FIG. 1. Plots of the logarithms of the dissociat,ion constants for
- -I
osnmate from the lactate dehydrogeuase-NAI>H-oxamate com-
600-
plex against reciprocal absolute temperatine (T). The measure-
ments were made in 0.1 M sodium phosphate, with ionic strength of
0.3 M with NaCl. I,ines were drawn by a least squares analysis.
l , pH G.00; A, pH 7.00; 0, pH 8.00.

l’he,,,rodgnunlics of oxawmle dissocinlion at 20’ in, 0.1 .XI sotli~r~~~

phosphate, with ionic si?ength of 0.3 .II wilh LVnCl

PII Ai? AII” AS” 2 4 6 8 IO

I
kcal/mole e.t1.
([Siteslfree + [Oxornate]) X IO5
G.00 6.37 zk 0.2oa 4.60 xk 0.25” -G.O zt 1.5”
7.00 6.54 xt 0.20 7.40 f 0.80 2.0 f 3.4 FIG. 3. Plots of the reciprocal relaxation times against concen-
8.00 6.15 f 0.20 12.1 + 0.7 20.3 f 3.1 trations for the system lactate dehydrogenase-NADH + oxamate
at 20”, 0.1 M sodium phosphate, with ionic strength of 0.3 M with
n Probable error. NaCl. Lines were drawn by the method of least squares.
4378 Lactate Dehydrogenase Vol. 244, No, 16

not always give a quantitative measureof helix content. The

value of X, isin goodagreementwith a value reportedby Jaenicke
(30) for the pig heart enzyme, and by Bolot,ina et al. (7) for the
pig skeletalmuscleenzyme.
Neither NADH nor oxamate, at concentrationssufficient to
saturate the binding sit,es,hasany detectableeffect on the optical
rot,atory dispersionspectrum of the protein, and no ext.rinsic
Cotton effects are observedin the 340 nm region of NADH ab-
sorption. The former resultsare in contrast with the findings of
Bolotina et al. (7), who reported large changesin bot’h boand X,
when NADH and oxalate are bound to pig skeletal musclelac-
tate dehydrogenase.
The observation t.hat b0and X, are essentiallyindependentof
pH in the region of neutrality corroborat,esJaenicke’smensure-
ments (30), but differs considerablyfrom results obtained with
pig skelet.almuscleenzyme (7).
Kinelics-The kinetics of NADH and oxamate binding to pig
350 400 450 500 heart lactate dehydrogenasecanin eachcasebe analyzed in terms
Wavelength, nm of a single relaxation process. The concentration dependences
FIG. 4. Difference absorption spectrum of pig heart lactate of the relaxation times are consistentwith singlestep, bimolecu-
dehydrogenase in 0.1 M sodium phosphate, with ionic strength of lar binding reactions (Figs. 2 and 3).
0.3~withNaCl,20”. Four l-ml cuvetteswith 0.5~cmpath lengths The difference spectrumobtained on TNAD binding (Fig. 4)
were used. Enzymesolutionswere 2.5 X ~O+J M in sites. TNAD was used to investigate the binding kinetics OFthis oxidized
n sampleand referencebuffer cuvettes was3 X 10-zM.
coenzyme to lactate dehydrogenase. In temperature jump
experiment.swith this molecule, two relaxation times can be
clearly discerned(Fig. 5). The shorter of these is observedin
t.heabsenceof enzyme, and is evident both at pH 6 and at pH 8.
Its estimated value is lessthan 3 Msec. The determination of
the actual relaxation time is instrument-limited. Even at 3”,
this relaxation effect is completewithin the heating t,ime of the
solution. There was no apparent effect on the velocity of this
reaction over a 20-fold range in coenzyme concentration.
The secondrelaxation phenomenon,which gives rise to the
larger optical density change,is associatedwith the binding of
coenzyme (Fig. 6).
The dissociation(kn) and recombination (kR) rate constants
for the various reactionscan be obtained from the plots of Figs.
2, 3, and 6. These values are summarizedin Table IV. The
ratios of theserate constants,kD:kR, are in goodagreementwith

Fro. 5. Relaxation experiment for the interaction of lactate 8000

dehydrogenase with TNAD in 0.1 M sodium phosphate, with ionic
strengt.hof 0.3 Mwit’h NaCl, 20°, at pH 6.00. [Sites] = 1.GX lo-*
sr; [TNAD] = 5.3 X W4 M; scanning rate, 0.1 msec per large di- L- 6000
vision; sensitivity, 20 mv per cm; total signal, 4 volts.
::
ments performed at two different temperatures (see below). 22 4000
The values obtained at pH 6 for the standard enthalpiesof dis- -t:
sociation of NADH and TNAD are, respectively, 13 and 9 kcal -
per mole. Becausethe data are limited, these estimatesare 2000
probably correct to within a factor of 2.
Optical Rotatory Dispersion-The optical rotatory dispersion
spectrum of pig heart lactate dehydrogenasehas the general 2 4 6 8 IO
features of a protein containing regions of a helix. These in-
clude a trough at approximately 233 nm, [01] = -6600”, and a
shoulder at about 215 nm, [Q] = 7600°. In addition, a small
@qree -
+ [TNAD]) x lo4

positive Cotton effect can be discernedin the region of aromatic FIG. 6. Plots of the reciprocal relaxation times against concen-
absorption. The fraction of cr helix, estimated from the bo trat.ions for the systemlactate dehydrogenase+ TNAD in 0.1 M
sodiumphosphate,with ionic st.rength of 0.3 M with NaCI, p1-I
values (Table III), is approximately 30%. This is only an order 6.00. Lines were drawn by the method of least squares. l , at
of magnitude assessment,since the Moffitt-Yang theory does 20”; 0, at 3”.
Issue of August 25, 1969 Heck 4379

Table IV such bonds couId easily va.ry with pH if the conformation of t.he
Rate contaits for binding reactions of pig heart lalate dehydrogen- active site were pH-dependent. The abnormal effect. of pH on
aee in 0.1 aodium phamphole, with inonic alongth of o s m with t,hc oxamate binding enthalpy might t.hus result from a pII-
dependent, conformational change at the active site. .-Z con-
NaCl, from temperature jump experiments
formational change in this region of pH has been suggested by
ko ke
Criddle el al. (34) from sedimentation velocity measurements.
WV The pH dependence of the entropy of oxamate dissociation,
0.96 Tk O.lTb given in Table IX, also indicates that this hypothesis is plausible.
NADH 0.25 + 0.14 The dat.a of this table show that increasing pH causes a large
0.70 i 0.05 increase in the entropy of oxamste dissociation. These data,
suggest that oxamate binding may “freeze out” one of several
17.2 f 0.9
OXAMITE possible protein conformations, a larger set of conformations
28.0 f 1.2
being available at pH 8 than at pH 6. Thus, the effec.t of in-
410 ik 60 creasing pH might be to loosen the structure of the prolein at
1.53 * 57 the active site. This would not be surprising, since t,he iso-
electric point of lactate dehydrogenase is in the acid region (33).
Since the optical rotatory dispersion spectrum does not vary
b ProbabIc erro, measurably with pH, all of the protein conformations involved
c =It, 3O. must have indistinguishable rotatory dispersion qxxtra.
d TIE concentration of free SADH n-as IO-folrl larger than Previous stopped flow measurements of the r:~te cotvtant for
&AD,.
SADH dissocia.tion from pig Hd lactate deh-drogcwase showd
i1~a.t this rate constant is independent of pH betn.een pH 6 and 8,
vnIucs for t,he dissoci:itinn consta.nls obtained by equilibrium and that, under basic conditions, KADH dissociation is the rate-
tit#r:ltions (Till1lC I). limiting step in enzymatic catalysis from the KU)+-lactate
side (10). These experiments also proved t.hat the mechanism
DISCUSSIOP;
of coenzyme replacement is dissociative, i.e. at high concentrn-
The rc~ults of the opt.&1 rotat,ory dispersion measurements tions of PJAD+ the replacement rate of ?jADH by KAD+ at the
can be summarized as f01l0~~r-s. (a) No new Cotton effects are ac.tivc site is independent of NAD+ concentration.
obserred when SXl)H and oxnmate are bound to l)ig hea~rl; IRC- The question of the actual mechanism of N-WI-I binding was
trite dehydrogeww; (0) no ci~anpc in the parameters X, or & not answered by the stopped flow experiments, bon-ever, since
for the protein are found when these molecules arc bound to the it. could not be asoertained from these experiments n-hethcr
enzyme; (3) there is no evidence of change in the helical content SXDH dissociation or a slow conformational change accompany-
betn-een pH 6 end pH S. ing that dissociation was, in fact, the siow step in the process of
The obser\+tllion that the optical rotatory dispersion speckurn cocna~me rcIAacemcnt.
of pig heart lacltlte dchydrogcnase is rlnchanged by KADH The relaxation experiments provide a direct answer to this
binding is in :>.greement, mith the findings of Listowsky el al. (31) question. The rate constants for KhDH dissociation, deter-
with bovine heart and rabbit muscle lactate dchpdrogenascs. mined bq’ u temperature jump method, are itlenticsl with the
WC have &ended these measurements to include osamate bind- dissociat,ion rate constants mea.sured by cocnzynw replacement
ing. The combination of these experiments shows no obscrva- in the stopped flow (10). Thus, thcsc results show that it, is the
ble chmge in the helica1 content of lactate dcbydrogenase when &woe&ion of h’hI3H that is the rate-limiting stcl), tind that a
complexed with NADH a,nd osamate. It should be noted that, slow conformational change does not occur (LI wry ral)id con-
in contrast, significant changes in the optical rotnt.ory dispersion formational change is, of course, still possible). The optical
parameters were found n-ith plb‘0. skeletal muscle lactate dehy- rotatory dispersion measurements indica.te that, as far as helical
drogenasc (7), Tvhich contains a considerable fraciion of 171 type content is concerned, no measurable structural changes take
subunits (32). The purity of the preparation of Uolotina et al. place.
(7) was not, honever, reported. ln contrast to the results reported here, C’zerIinski and Schrcck
The ~1-1 dependence of the oxamate binding enthalpg is of (S) reported that the binding of NADH to rabbit: muscle lactate
interest. If t.he interaction of oxama,te with t.he protein were dchydrogcnase, which consists almost exclusively of the LJd
purely elect.rost.atic, one might, expect that incrcaaing the pH isoenzyme (35), occurs in at least two steps, one of which is RI)-
would lead to decreased enthalpies of osa.nlnle dissociation, pnrently a structural rearrangement. The various relaxation
since the total chsrgc on the protein would become more nega- experiments therefore attest to different mechanisms of SADH
t,ivc. Ex;lctly t.hc reverse is found, however. This unexpected binding to I-1 and R/I subunits, in agreement with other data
effect of pH on the binding enth&g is so large tha.t at low tem- (3, 4, 6).
perat.ures (2’ < 13”) ox&mate is actually bound more Oightlg at The dissociation ra.te constant for TSAD is considerably
pH S than at pH 6 (Fig. 1). It is evident, therefore, t,hat a la.rger than i’or XADH, whereas the recombination rate constant,
simple electrostatic int.eraction is insufficient to account for is smaller for the oxidized coenzyme. Velick (25) has suggested
osamnte binding. a reasonable mechanism to account for the differences in the
Alfhough electrostatic forces a.pparentl- arc of major impor- affinities of lactate dehpdrogenase for oxidized and reducrd coen-
tance in inhibitor binding (a-1), it is reasonable to assume that ZJ-mcs. According to this vieK, the effect of osidizing S;IDH is
other types of bonding, probabljd hydrogen bonding, arein\-old-ed to cause a change in bond angles on the nitrogen of the pyridinium
in the interaction of osanmte with the protein. The strength of ring. This would result in a conformational adjustment of the
4380 Lactate Dehydrogenase Vol. 244, No. 1G

entire dinucleot,ide, leading to weakened interactions with the lapsed form, with overlap of pyridine and adenine rings. 111
protein at possibly several points. addition, Czerlinski and Hommes (39) reported t,hat bot,h NXDH
It is not difficult to see how the weak forces of enzyme-sub- and NADPH undergo temperature-dependent, isomerizations in
strate complex formation, many of which are short range (e.g. solution, which occur in the 100 psec range. Thus, the present
van der Waals-London forces, hydrogen bonds), could have a experiments are in agreement with the postulate of Jardetzk)
large effect on the rate constants of a “diffusion-controlled” and Wade-Jardetzky (38) and indicate that the relaxat,ion time
reaction. The reaction of lactate dehpdrogenase with coenzyme for the oxidized form is considerably shorter than for the reduced
may be formally written molecule. It is presumably only the elongated form which is
bound to the enzyme (38).
k12 ha The kinetics of osamatc binding provides rvidcnce that IIO
E+C F==-+ E...C \\ EC
km kaz (1) slow relaxation effects occur during oxamate binding. Kecrllt
(1) (2) (3) esperiments have shown, however, that during pyruvate binding,
the initial enzyme-NADIR-pyruvate complex undergoes a l)II-
where (2) is an encounter complex dependent rearrangement to a catalytically active ternary com-
If the intermediate encounter complex is assumed to be in a plex, which is sufficiently slow to be measurable, in the stopped
steady state, there are two limiting cases for the diffusional rate flow (34). It is difficult to conceive of a better nonreactive
constants (36): (n) the diffusion step (1-2) is rate-limiting, i.e. structural analogue of pyruvate than is oxamatc. Nevertheless,
k23 >> k21. Then the latter is evident,ly un:tble to induce a similar collformatioll:~l
change in the protein.
kn = kl?; The kinetic data provided in this paper and elsewhere (10, 34)
arc of significance for underst,antling the mechanism of catalysis
(b) The transformation step (2-S) is rate-limiting, i.e. kZ3 << ktl. by rnzymes such as lactate dchydrogennse. &cause this cn-
Then z\-me, like several other dehydrogcnases, has a compulsory bind-
ing order (28), it is important that the velocities of the initial
kl2 steps be known and their mechanisms understood. The mecha-
kR = - . kz; kD = x.31 (3)
kz, nisms of NADH binding to livrr alcohol dehydrogenase (40), pig
heart, mnlate dehydrogenase (41), and rabbit muscle lactate
For the explicit calculation of the rate constants from diffusion
dehydrogenase (8) have already been studied by stopped flow
theory, Case a must be assumed.
or temperature jump techniques. Kinetic evidence for :I co-
If, however, Case b holds, the short range forces become
enzyme-induced structural isomerization has been reported 0111~
extremely important. Assuming that k 1%and kZl arc identical
in the case of the rabbit muscle enzyme. Although the presrnt
for TShD and XhI)H, we have from Equation 3 :m(l Table IV
results agree satisfact,orily with simple schemes for coenzymc
that,, at 20” and pH 6,
binding, they do not, of course, exclude the possibility of ver?
kR, NADH k?z, NADH rapid structural rearrangements nor, in the case of TNhl), of
=--=G (4) slow conformational changes with small amplitudes.
kR , TNAI) kza, TN.411

and :lcknowlecZgnrenfs-I would like to thank Dr. Manfred Eigcn

ko, NAUH kaZ, NADH in whose laboratory the majorit,y of these experiments was Ijer-
= 0.013 formed, rind Dr. (‘. T. O’Konski for a stimulating discussion.
kD, TNAD = ktz, TNAl1
REFERENCES
Such effects are readily conceivable in terms of short range
1. L.\TNER, A. 1, ., ANI) SKILLEN, A. W., IsoenztJnles in bio!ogU and
forces. It is plausible that such forces would strongly influence ?nedicine, Academic Press. New York. 1968.
ka2but have a relatively small effect o11&. The model proposed 2. C.\HN, 1~. I>., KAPL.\N, N. d., LEVINE, i., .\NI) %~ILLISG, E.,
by Velick (25) is thus consistent with the kinetic data which are Science,136, 9G2 (1962).
obscrvetl. t7 ZE\VB, V., .4x1) FIIOMM, H. J., Biochemislq, 4, 782 (1965).
4. Nova.\, W. IS., WINER, A. I)., GILAID, A. J., .\NI) SCH\\.ERT, G.
Three possible hypotheses could explain the rapid chemic~:rl W., J. Biol. Chem., 234, 1143 (1959).
relaxation which is found with TNAD. One mechanism would 5. ROSSM.\NN, M. G., .JEFFEKY, 13. A.! M.+IN, I’., .~uL) W.\IU<ES, S.,
involve a fast protonation-deprotonation equilibrium in the cocn- Proc. Asaf. Acutl. Sci. U. S. A., 67, 515 (1967).
zyme. That this explanation is iml)lnusible is indicated by the G. ANDERSON, S. I<., .\NU WEI~EI~, G., Biochemis/ry, 4,1948 (1965).
following. (a) NAI)+ does not have a pK in the $1 range in- 7. BOLOTIN.L I. A.. M.UZKOVICH. D. S.. VOLKENSTEIN. M. V.. .w)
ZAVODZI~Y, I’.; Biochim. Bioph,ys.‘Acta, 132, 271 i19G7).’
vestigated (37), and (b) the amplitude and velocity of the effect 8. CZERLINSKI, G. II., .\NI) SCHRECK, G., J. Biol. (‘hem., 239, 913
are not pert,urbed by changing the pH from 6 to 8. (1964)
A second possible explanation is that the effect, is a bimolecular 9. HATH.ZN~~LY, G., .LND GRIDDLE, I<. S., Proc. iTat. Acntl. Sci.
binding reaction between 2 molecules of coenzyme. The obscr- U. S. A., 66, 680 (1966).
10. HECK, H. D’A., McMun~t.\~, C. II., ;\NI) GUTFILEL~ND, H., Bio-
vation that this effect is independent of coenzyme concentration them. J., 108, 793 (19G8).
renders this explanation unacceptnblc. 11. 131 SABATO, Cr., AND OTTESEN, M., Biochemislry, 4, 422 (1965).
The most reasonable explanation, therefore, is that TNAD 12. WIEL.IND, T., .\NI) PFLEII)ERER, G., Anger. (‘hem. Inf. Ed.
undergoes a temperature-dependent structural isomerization in Engl., 1, 169 (19G2).
solution. Evidence in support of this hypothesis has ~XTII ob- 13. HOLBROOK, J. J., PYLEIDERER, G., SCHETOEK, J., AND 111~
MIIIR. S.. Biochem. Z., 344, 1 (1966).
tained by Jardetzky and Wade-Jardetzky (38), who postulated 14. VESELS;, E’. S., AND YI&LUI~C, k. i., Proc. .Vaf. Acad. Sci.
two conformational forms of Nr\D+, an elongated form and a col- U. S. A., 56, 1317 (1966).
.[ssue of August 25, 1969 H. d’A. Heck

15. J.\ENICKE, Ii., .\ND KNoF, S., Eur. J. Biochem., 4, 157 (1968). 29. SCH~EI~~~, G. W., MILLER, B. R., AND PEANASKY, R. J., J. Biol.
10. WXIISMUTII, E. n., AND PFIZIDEIZER, G., Biochem. Z., 336, Chem., 242, 3245 (1967).
545 (1963). 30. JAENICICE, R., Biochim. Biophys. Acta, 86, 186 (1964).
17. WIEI,.\ND, T., I)~Es~EI~G, I’., PFLEIDERER, G., STOCK, A., AND 31. LISTO~SKY, I., FURFINE, C. S., BETHEIL, J. J., AND ENGLARD,
SANN, E., Arch. Biochem. Biopkys., Suppl. 1, 260 (1962). S., J. Biol. Chem., 240, 4253 (1965).
IS. EIGEN, M., ANI) DE M~EYEIL, L., Tech. Org. Chem., 6, 895 (19F3). 32. MARI~ERT, C. L., AND MOLLER, F., Proc. Nat. Acad. Sci. U. S.A.,
19. Ilo~u~~oo~c, J. J., Biochem. Z., 344, 141 (1966). 46, 753 (1959).
20. Y.\NG, J. T., AND DOTY, P., J. Amer. Chem. SOL, ‘79, 761 (195i). 33. JAENICKE, It., AND PFLEIDERER, G., Biochim. Biophys. Acta,
21. MOFFITT, W., AND YI\NG, J. T., Proc. Xaf. Acad. Sci. U. S. A., 60, 615 (1962).
42, 596 (1956). 34. CRIDDLE, R. S., MCMURRAY, C. H., AND GUTFREUND, H.,
22. WACHSMUTH, E. I)., PPI,EIDEREI%, G., AND WIELAND, T., Bio- Nature, 220, 1091 (1968).
them. Z., 340, 80 (1964). 35. WIELAND, T., PFLEIDERER, G., HAUPT, I., AND WOERNER, W.,
23. WINEI<, A. I>., SCHWERT, G. W., AND MILIAI~, n. B. S., J. Rio/. Biochem. Z., 332, 1 (1959).
C’hem., 234, 1149 (1959). 36. EIGEN, M., Z. Phys. Chem., N. F., 1, 176 (1954).
24. WINER, A. l)., AND SCH\I.ERT, G. W., J. Biol. Chem., 234, 1155 37. VON EULER, H., AND SCHLENIC, F., Hoppe-Seyler’s Z. Physiol.
Chem., 246, 64 (1936).
(1959).
38. JARDETZI~Y, O., AND WADE-JARDETZICY, N. G. W., J. Biol.
25. VEI.ICI~, S. F., J. Biol. Chem., 233, 1455 (1958).
Chem., 241, 85 (1966).
26. FItoMnf, II. J., J. Biol. Chem., 238, 2938 (1963).
30. CZERLINICSI, G., AND HOMMES, F., Biochim. Biophys. Acta, 79,
27. McK.\y, 11. H., AND KAPIAN, N. O., Biochim. Bioph!/s. Acta, 46 (19G4).
‘79, 273 (1964). 40. GERACI, G., AND GIBSON, Q. H., J. Biol. Chem., 242, 4275
28. TA~~ENAICA, Y., .\ND SC~I\YEI~~~, G. W., J. Biol. Chem., 223, 157 (1967).
(1956). 41. CZERLINSICI, G. H., AND SCHRECI~, G., Biochemistry, 3, 89 (19F4).