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Studies of enzyme kinetics
atRCHIVF,S
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Studies il. Inhibition
of Pyruvic
94,
236240
of Enzyme
Kinetics
Carboxylase Pyruvic Acid’
by Certain
GLEN From
(1961)
Analogs
of
R. GALE2
the Veterans Administration Hospital, and the Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina Received
January
23, 1961
A series of structural analogs of pyruvic acid were tested as possible inhibitors of pyruvic carboxylase. It was found that substitutions on the p-carbon atom yielded the most potent inhibitory compounds. A kinetic study of five such inhibitors indicated a progressive irreversible inhibition, preceded in some cases by an immediate reversible phase. Inhibition was apparently mediated at the substrate-binding site. METHODS
INTRODUCTION
As one of a series of investigations of inhibition of enzymes unique in microorganisms by substrate analogs, pyruvic carboxylase was chosen due to the scarcity of data pertaining to its inhibition. Green et al. (3)) using a highly purified carboxylase, demonstrated inhibition by silver, copper, or mercury ion, whereas acetaldehyde had only a slight depressing effect and iodoacetate, lactate and arsenate had no effect. Watt and Werkman (5) showed that the enzyme in Aerobacter aerogenes and yeast maceration juice was inhibited by phenylpyruvate, and Redemann and Meikle (4) reported that 2,2-dichloropropionic acid was a competitive inhibitor at low concentrations and an uncompetitive one at higher concentrations. The following is a report of the effect of certain other struct’ural analogs of pyruvate on the yeast carboxylase. ‘Aided by Grant E-861, National Microbiological Institiltc, National Institutes of Health, Department of Health, Education, and Welfare. ‘With the technical assistance of Randall I,. Harrington and Ann Marie Welch.
Pyruvic rarboxylase was partially purified from Fleischmann’s type 20-40 dried yeast (Standard Brands) by the method of Green et aE. (3). After step three of their method, the precipitate was dissolved in 200 ml. of 0.04 M citrate buffer at pH 5.9, 76 g. (NHa)SO, was added, and the resulting suspension was stored at -10°C. For each experiment an nliouot of this was centrifuged at 10,000 X g for 10 min. at 5”C., the supernatant solution decanted, and the precipitate dissolved in 0.04 M citrate buffer, pH 5.9, so that 1.0 ml of the resulting enzyme solution resulted in an initial velocity of approximately 17 ~1. COz/min. from 20.0 pmoles pyruvic acid (Na salt, Sigma) in a reaction volume of 2.2 ml. at 30°C. and pH 5.9 with 0.04 M citrate buffer. In assays of each batch of enzyme purified, a linear relationship between v and enzyme concentration with intercept at the origin was noted, indicating no inhibit,ors or activators in the preparations which would influence the results during the test period. There was no decrease in activity upon storage at -10°C. Bnalogs of pyruvic acid obtained from commercial sources were : acetamide, ncctyluren, a-bromopropionic acid, a-chloropropionic acid, oxnnilic acid, o-nitrophenylppruvic acid, and 2,3-butanedione (Eastman); ethyl pyruvatc and ,% chloropyruvic acid (Bias) ; propionic acid (Fisher) ; phenylalanine (Calif. Corp. for Biochemical Research); oxamic acid (Matheson, Coleman and 236
IWZTME
KINETICS.
Bell) ; oxalacetic acid (Krishell) ; oxalic acid (iM:dlinckrodt) ; sodium phenylpyruvnte and DL/%phenyllactic: acid (ISutritional Biochemicals) ; p-hydroxyphcnplpyr~l~~i~ arid, gloxylic acid, and kctomalonic acid (HM) ; and a-ketoglutaric acid (Sigma). All compounds were soluble in ritrate buffer at the cwwcnt,rat,ions usad, and the pH of eac.11 solution was adjusted as necessary. Mwsuremc~nts of enzyme activity were done with the Warburg rcspirometcr at 30°C. in vessels of approximately 15 ml. wpncit,y. The atmosphere in the vessels wiis air. Initial velocity, L’, was tletcrmined by plotting log ((1 - 2) against time in minutes, where n = initial pyruvate concentration exI,ressed as ~1. CO, which would br: produced upon complete decarboxylation (= pmoles X 22.4), and z = ~1. CO, produced at any given time. From this, the regression coefficient was calculated, which, when multiplied by the factor for conversion to natural logarithms, was I;, the velocity constant of the reaction. The initial velocity was then computed by multiplying 12 by the initial subatratc c~onwntration, 0; 2) was espressetl as ~1. COJmin. In all (715es control vessels with no 20 min. drllg resulled in a st,raiglrt, line for at, lf7& when log (a - 2) was plotted against time, indicating first-o&r kinetics. This was also true in the I>rwenccJ of the proposed inhibitors, lvith the exception of 2,3-but anctlione. With this a slowing of the velocity ocwrred :rftc,r 10 min.; therefore, only readings during Ihc first 10 min. wcrc wed in wlculating /‘. RRSVLTS
AYTD DISCUSSION
Of the 20 compountl~ testetl, inhibition was obtained usually with those xvhich cont’ained the basic three-carbon fragment of pyruvate with an unaltered a-kctocarboxylic group, i.e., ,8-substituted pyruvate (Table I). These were p-chloropyruvate, I’hc’nyll’yruv~~tc, o-nitrophenylpyruvate, juhydroxyphenyll)yru~rate, ketomalonate, CLketoglutarato ant1 glyoxylatc. The only excc~ptions to this were oxanilic acid, a weak inhibitor, which rcscinblts phenylpyruvatc, a nitrogen atom having been substituted for the p-carbon atom, ant1 2,3-butanedione. The only ,6!-substituted compound tested which failed to show inhibition wis oxalacetrite. Substit,ution of the kcto oxygen by two hydrogen atoms (propinnate) or one hydro-
237
II TABLE
INHIBITIOS
OF THE
CARROSYLASE Suhstjr:ttc
IXITIAL BY
I ~ELCKXTB
concentjration
OF PYRVVIC
A~ars~c,s
SCBSTRATE 10.0
pmoles/2.%
ml.
(= 1.5 X 10m3 M) throtlghout. Inhibitors jvere added to t.he c~myrnc 15 min. before addition of sutx3tr:ttr. analog
Molar
concentration
Inhibition
cp-Hydrosypherl~lpyruvate p-Hydroxypherlyll,yruvate
4.5 x to--*
f2
1.1 X 10-z
71
Chloropyruvate Chloropyruvate
4.5 2.3
Glyoxylate Glyoxylate
2.3 x IO-” 4.5 x IO-”
33
o-Nitrophenylpyruvate o-i%trophenylpyruvate
9.0 x 10-j I.4 x 10-d
Gl
Ketomnlonate Ketomslonate
2.3 x 10-J 3.1 x IO--’
8X
Phenylpyruvate Phenylpyruvate
1.1
08
2.3 x lo-”
80
Osanilat~e
0.0 x IO-”
31
2,3-Uiitanedione
9.0 x IO-3
56
a-Ketoglutarate
2.7
2-l
x
lo--I
27
x
10-3
T3
x
x
lOY3
10-2
95
70
71
gen and one halogen atom (a-chloro- or UI)ronloI’roI)ion;te) failed to yield inhibitory coml)ounds. Substit’ution of this oxygen atoiil by one hydrogen atom and one amino group, even with a phenyl group on the ,#-carl)on atoln I plienylalanine) had no inhibitory effect on the enzyme. Similarly, reduction of phcnylpyruvatc tJo phenyllacMe resulted in :t noninhibitory compound. Subst,itutions of the carboxyl carbon or the p-carbon atom by amino groups (acetamide and oxanwtc, respectively), the former by urw I acc~tyhuw~, or the 1;ttt.w by a hyclroxyl group (osalatc) gave compounds complrtcly without inhibitory activity. Convcwion of I)yruvatt: to the ethyl ester yielded an inactive con-~pound.
238
GALE KINETICS
OF INHIBITION
Of the compounds which were inhibitory, five true p-substituted analogs were chosen for a kinetic study of the inhibition. Weak inhibitors (oxanilate, a-ketoglutarate) were omitted, as were /3-chloropyruvate due to difficulties encountered in obtaining it in crystalline form, and 2,3-butanedione due to the progressive inhibition with time displayed with this compound. In experiments in which phenylpyruvate, o-nitrophenylpyI/S II I03 glyoxruvate, p-hydroxyphenylpyruvate, FIG. 1. Reciprocal plot to show typical nonylate, or ketomalonat’e was added to the competitive inhibition of pyruvic carboxylase of main compartments of the Warburg vessels the type obtained with each of five analogs of and pyruvate tipped in from the side arms 10 pyruvate. Concentration of inhibitor (o-nitromin. later after temperature equilibration, phenylpyruvate), 4.5 X lOa M. Velocity is pl. it was found that each compound gave reCOJmin.; substrate concentration is ~1. CO2 which sult,s compatible only with noncompetitive would be evolved upon complete decarboxylation inhibition. A typical experiment is plotted in a final volume of 2.2 ml. The lines are leastin Fig. 1. This indicated either combination squares lines. of the inhibitor with some site other than the active substrate-binding site, or an irreversible combination at the substratebinding site. Consequently, experiments were conducted in which each inhibitor was added along with the pyruvate, and 15 min. prior to pyruvate. Results are shown in Fig. 2. In each case simultaneous addition of substrate protected the enzyme from the inhibitor, up to 100% in the case of ketomalonate and p-hydroxyphenylpyruvate, indicating that the inhibitor was bound at the substrate-binding site, a situation compatible with the structures of the inhibitors. Furthermore, there appeared to be no inhibition becoming evident in those vessels in which substrate and inhibitor were added simultaneously, as manifest by the strict linearity of the plots of log (a - x) against time ; that is, the presence of substrate prevented any apparent progressive inhibition. MINUTES Dixon and Webb (2) state that a reversibly FIG. 2. The course of decarboxylation of pyrucombining substrate can never prevent the vate by pyruvic carboxylase with ( l ) no inhibitor ultimate complete inhibition by an irreverspresent, (X) inhibitor added 15 min. before pyible inhibitor, so long as sufficient inhibitor ruvate, and (0) inhibitor added simultaneously is present to saturate the enzyme, since with pyruvate. Inhibitors, with final molar concenthere is always a small amount of free entrations: A, phenylpyruvate, 4.5 X lo-*; B, ketozyme as a result of the reversible equilibmalonate, 9.0 X lo-‘; C, o-nitrophenylpyruvate, rium with the substrate. This free enzyme 4.5 X 10-5; D, p-hydroxyphenylpyruvate, 4.5 X 10m4; is thus subject to the effects of the inhibiE, glyoxylate, 3.2 X 10T6. Pyruvate concentration, 4.5 X lo-’ M. The lines are least-squares lines. tor. The above mentioned linearity of the
ENZYME
KINETICS.
plots of log (a - 5) against time is not necessarily a contradiction of this statement; rather, the period of measurement and experimental error of the method COUplod with the relative affinities of the enzyme for each of the compounds likely rcsulted in inability to detect the progressive inhibition. Since irreversible inhibition is generally characterized by a progressive increase with time, it was considered of interest to determinc the degree of inhibition produced by incubating the enzyme with inhibitor for varying periods of time before addition of substrate. Double side-arm vessels were set up so that each inhibitor could be added t,o the enzyme at precise times from one side arm before addition of substrate from the other. Figure 3 shows the results expressed as logarithm of velocity (= logarithm of enzyme remaining) after preincubation for specific intervals. A logarithmic plot is used t,o show the similarity to a first-order reaction, Thermal inactivation of the enzyme was not a factor in these experiments since all vessels were incubated for an equal time at 30°C. regardless of the interval between addition of inhibit,or and subst,ratc. To determine whether any of these five inhibitors was itself decarboxylated, 2.0 +olcs (45 ~1.) was added from the side arms of vessels containing three times the usual enzyme concentration. At the end of 2 hr. CO2 evolved from each was as follows: glyoxylate, 0; o-nitrophenylpyruvate, 0; kctomalonatc, 0; p-hydroxyphcnylpyruvate, 37 ,J.; phenylpyruvatc, 25 PI. A plot of log ( 0 - .r) against time for the two which lvere decarboxylatetl showed a decreasing velocity with t’imc. Inhibit,ion kinetics of the fire analogs so studied bear :I striking similarit’y to the kinetics of inhibition of specific acetylcholine&erase by certain organic phosphates ( I). Even though thesc latter compounds do not appear to be structural analogs in the general scns:c~,the mechanism of their inhibition of certain estcrases is actually a first-stage hydrolysis, the resulting phosphorylatetl enzyme being quite inactive (2). Xltlridge (1)) working with p-nitrophenyl tlic%hyl thiophosphatc, p-nitrophenyl rii-
0
239
II
5
IO
0 5 MINUTES
IO
0
5
IO
FIG. 3. Progressive decreasein velocity of pyruvic carboxylase by structural analogs of py-
ruvnts when inhibitors were added at certain intervals prior to addition of substrate. Inhibitors, with final molar concentrations: A, phenylpyruvate, 9.0 X 10e4; B, ketomalonate, 9.0 X 10A5; c, o-nitrophenylpyruvate, 4.5 X 10e5; D, p-hydroxyphcnylpgruvate, 4.5 X lo-‘; E, glyoxylate, 1.8 X lOmE.Pyruuate concentration, 4.5 X 10e3 M. The arrows indicate velocity in absence of inhibitor. The lines are least-squares lines.
ethyl phosphate, and S-quinolyl diethyl thiophosphate, found that when either of the first. two of these compounds was added to his cholincsterase preparations at certain times before addition of substrate, a plot of logarithm of percentage activity (= logarithm of velocity) against time of incubation yieldetl a straight line which intercepted the origin. When the quinolyl derivative was used, a straight line again resulted which! howcvcr, did not intercept the origin. Such results wit’h this latter compound led him to deduce that the difference between logarithm 100% activity and the actual intcrccpt of the experimental plot w-as :I measure of reversible inhibition; the progressirc linear portion of the plot was the irreversible portion. ,4n analogous situation appears evident with carboxylase inhibitors. Phenylpyruvatc, o-nitrophenylpyrurate, and glgoxylate each yielded a progressive inhibition which did not intercept the origin. Thr former ~v-ns the only inhibitor the effects of which were partly rcverscd by dialysis. Clpoxylate, phcnyl-
240
GALE
pyruvate, and its o-nitro derivative thus appeared to yield kinetic results similar to the 8-quinolyl derivative of Aldridge (1) while the other two were more analogous to his p-nitrophenyl derivatives. On the basis of the experimental data presented, an exact mechanism of inhibition cannot be inferred. The evidence indicates t’hat the five analogs studied in most detail combine with the active site of t,he enzyme. The progressive inhibition occurring before addition of substrate could be the result of simply a slow uptake of the inhibitor, the final product being a complex between the enzyme and unaltered analog. Since, however, decarboxylation of two of these apparently occurred, the true inhibitor may have been the aldehyde corresponding to each of the analogs. The fact that decarboxylation of ketomalonate, glyoxylate, or o-nitrophenylpyruvate could not be demonstrated does not necessarily invalidate such a proposal since the aldehydes theoretically thus formed were perhaps more potent inhibitors than the others and the total amount of COZ evolved before complete inactivation of the enzyme could well have
escaped detection due to limitations of the method. The likelihood that a nondissociable bonding occurs between any such aldehydc formed and the enzyme as with inhibition of choline&erase by the organic phosphates seems somewhat, remote. If this were the cast, at any given enzyme concentration the dcgrec of inactivation of the enzyme would be the same upon decarboxylation of a certain number of molecules of any of the inhibitors. The fact that p-hydroxyphenylpyruvate was almost completely decarboxylated at the concentration employed, the velocity decreasing with time, suggests rather that each molecule of aldehyde, if formed, partially inactivates the active site at which it was formed. REFERENCES 1. 2.
J. 46, 451 (1950). C., “Enzymes.” ,4cademic Press, New York, 1958. 3. GREEN, D. E., HERBERT, D., .~SD SUBRAHMANYAN, V., J. Bid. Chem. 138,327 (1941). 4. REDEMANK, C. T., AND MEIKLE, R. W., Arch. Biochem. Biophys. 59, 106 (1955). 5. WATT, D. D., ASD WERKMAN, C. H., Arch. Biothem. Biophys. 50,64 (1954). ALDRIDGE, W. K., Biochem. DIXON, M., AND WEBB, E.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Studies il. Inhibition
of Pyruvic
94,
236240
of Enzyme
Kinetics
Carboxylase Pyruvic Acid’
by Certain
GLEN From
(1961)
Analogs
of
R. GALE2
the Veterans Administration Hospital, and the Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina Received
January
23, 1961
A series of structural analogs of pyruvic acid were tested as possible inhibitors of pyruvic carboxylase. It was found that substitutions on the p-carbon atom yielded the most potent inhibitory compounds. A kinetic study of five such inhibitors indicated a progressive irreversible inhibition, preceded in some cases by an immediate reversible phase. Inhibition was apparently mediated at the substrate-binding site. METHODS
INTRODUCTION
As one of a series of investigations of inhibition of enzymes unique in microorganisms by substrate analogs, pyruvic carboxylase was chosen due to the scarcity of data pertaining to its inhibition. Green et al. (3)) using a highly purified carboxylase, demonstrated inhibition by silver, copper, or mercury ion, whereas acetaldehyde had only a slight depressing effect and iodoacetate, lactate and arsenate had no effect. Watt and Werkman (5) showed that the enzyme in Aerobacter aerogenes and yeast maceration juice was inhibited by phenylpyruvate, and Redemann and Meikle (4) reported that 2,2-dichloropropionic acid was a competitive inhibitor at low concentrations and an uncompetitive one at higher concentrations. The following is a report of the effect of certain other struct’ural analogs of pyruvate on the yeast carboxylase. ‘Aided by Grant E-861, National Microbiological Institiltc, National Institutes of Health, Department of Health, Education, and Welfare. ‘With the technical assistance of Randall I,. Harrington and Ann Marie Welch.
Pyruvic rarboxylase was partially purified from Fleischmann’s type 20-40 dried yeast (Standard Brands) by the method of Green et aE. (3). After step three of their method, the precipitate was dissolved in 200 ml. of 0.04 M citrate buffer at pH 5.9, 76 g. (NHa)SO, was added, and the resulting suspension was stored at -10°C. For each experiment an nliouot of this was centrifuged at 10,000 X g for 10 min. at 5”C., the supernatant solution decanted, and the precipitate dissolved in 0.04 M citrate buffer, pH 5.9, so that 1.0 ml of the resulting enzyme solution resulted in an initial velocity of approximately 17 ~1. COz/min. from 20.0 pmoles pyruvic acid (Na salt, Sigma) in a reaction volume of 2.2 ml. at 30°C. and pH 5.9 with 0.04 M citrate buffer. In assays of each batch of enzyme purified, a linear relationship between v and enzyme concentration with intercept at the origin was noted, indicating no inhibit,ors or activators in the preparations which would influence the results during the test period. There was no decrease in activity upon storage at -10°C. Bnalogs of pyruvic acid obtained from commercial sources were : acetamide, ncctyluren, a-bromopropionic acid, a-chloropropionic acid, oxnnilic acid, o-nitrophenylppruvic acid, and 2,3-butanedione (Eastman); ethyl pyruvatc and ,% chloropyruvic acid (Bias) ; propionic acid (Fisher) ; phenylalanine (Calif. Corp. for Biochemical Research); oxamic acid (Matheson, Coleman and 236
IWZTME
KINETICS.
Bell) ; oxalacetic acid (Krishell) ; oxalic acid (iM:dlinckrodt) ; sodium phenylpyruvnte and DL/%phenyllactic: acid (ISutritional Biochemicals) ; p-hydroxyphcnplpyr~l~~i~ arid, gloxylic acid, and kctomalonic acid (HM) ; and a-ketoglutaric acid (Sigma). All compounds were soluble in ritrate buffer at the cwwcnt,rat,ions usad, and the pH of eac.11 solution was adjusted as necessary. Mwsuremc~nts of enzyme activity were done with the Warburg rcspirometcr at 30°C. in vessels of approximately 15 ml. wpncit,y. The atmosphere in the vessels wiis air. Initial velocity, L’, was tletcrmined by plotting log ((1 - 2) against time in minutes, where n = initial pyruvate concentration exI,ressed as ~1. CO, which would br: produced upon complete decarboxylation (= pmoles X 22.4), and z = ~1. CO, produced at any given time. From this, the regression coefficient was calculated, which, when multiplied by the factor for conversion to natural logarithms, was I;, the velocity constant of the reaction. The initial velocity was then computed by multiplying 12 by the initial subatratc c~onwntration, 0; 2) was espressetl as ~1. COJmin. In all (715es control vessels with no 20 min. drllg resulled in a st,raiglrt, line for at, lf7& when log (a - 2) was plotted against time, indicating first-o&r kinetics. This was also true in the I>rwenccJ of the proposed inhibitors, lvith the exception of 2,3-but anctlione. With this a slowing of the velocity ocwrred :rftc,r 10 min.; therefore, only readings during Ihc first 10 min. wcrc wed in wlculating /‘. RRSVLTS
AYTD DISCUSSION
Of the 20 compountl~ testetl, inhibition was obtained usually with those xvhich cont’ained the basic three-carbon fragment of pyruvate with an unaltered a-kctocarboxylic group, i.e., ,8-substituted pyruvate (Table I). These were p-chloropyruvate, I’hc’nyll’yruv~~tc, o-nitrophenylpyruvate, juhydroxyphenyll)yru~rate, ketomalonate, CLketoglutarato ant1 glyoxylatc. The only excc~ptions to this were oxanilic acid, a weak inhibitor, which rcscinblts phenylpyruvatc, a nitrogen atom having been substituted for the p-carbon atom, ant1 2,3-butanedione. The only ,6!-substituted compound tested which failed to show inhibition wis oxalacetrite. Substit,ution of the kcto oxygen by two hydrogen atoms (propinnate) or one hydro-
237
II TABLE
INHIBITIOS
OF THE
CARROSYLASE Suhstjr:ttc
IXITIAL BY
I ~ELCKXTB
concentjration
OF PYRVVIC
A~ars~c,s
SCBSTRATE 10.0
pmoles/2.%
ml.
(= 1.5 X 10m3 M) throtlghout. Inhibitors jvere added to t.he c~myrnc 15 min. before addition of sutx3tr:ttr. analog
Molar
concentration
Inhibition
cp-Hydrosypherl~lpyruvate p-Hydroxypherlyll,yruvate
4.5 x to--*
f2
1.1 X 10-z
71
Chloropyruvate Chloropyruvate
4.5 2.3
Glyoxylate Glyoxylate
2.3 x IO-” 4.5 x IO-”
33
o-Nitrophenylpyruvate o-i%trophenylpyruvate
9.0 x 10-j I.4 x 10-d
Gl
Ketomnlonate Ketomslonate
2.3 x 10-J 3.1 x IO--’
8X
Phenylpyruvate Phenylpyruvate
1.1
08
2.3 x lo-”
80
Osanilat~e
0.0 x IO-”
31
2,3-Uiitanedione
9.0 x IO-3
56
a-Ketoglutarate
2.7
2-l
x
lo--I
27
x
10-3
T3
x
x
lOY3
10-2
95
70
71
gen and one halogen atom (a-chloro- or UI)ronloI’roI)ion;te) failed to yield inhibitory coml)ounds. Substit’ution of this oxygen atoiil by one hydrogen atom and one amino group, even with a phenyl group on the ,#-carl)on atoln I plienylalanine) had no inhibitory effect on the enzyme. Similarly, reduction of phcnylpyruvatc tJo phenyllacMe resulted in :t noninhibitory compound. Subst,itutions of the carboxyl carbon or the p-carbon atom by amino groups (acetamide and oxanwtc, respectively), the former by urw I acc~tyhuw~, or the 1;ttt.w by a hyclroxyl group (osalatc) gave compounds complrtcly without inhibitory activity. Convcwion of I)yruvatt: to the ethyl ester yielded an inactive con-~pound.
238
GALE KINETICS
OF INHIBITION
Of the compounds which were inhibitory, five true p-substituted analogs were chosen for a kinetic study of the inhibition. Weak inhibitors (oxanilate, a-ketoglutarate) were omitted, as were /3-chloropyruvate due to difficulties encountered in obtaining it in crystalline form, and 2,3-butanedione due to the progressive inhibition with time displayed with this compound. In experiments in which phenylpyruvate, o-nitrophenylpyI/S II I03 glyoxruvate, p-hydroxyphenylpyruvate, FIG. 1. Reciprocal plot to show typical nonylate, or ketomalonat’e was added to the competitive inhibition of pyruvic carboxylase of main compartments of the Warburg vessels the type obtained with each of five analogs of and pyruvate tipped in from the side arms 10 pyruvate. Concentration of inhibitor (o-nitromin. later after temperature equilibration, phenylpyruvate), 4.5 X lOa M. Velocity is pl. it was found that each compound gave reCOJmin.; substrate concentration is ~1. CO2 which sult,s compatible only with noncompetitive would be evolved upon complete decarboxylation inhibition. A typical experiment is plotted in a final volume of 2.2 ml. The lines are leastin Fig. 1. This indicated either combination squares lines. of the inhibitor with some site other than the active substrate-binding site, or an irreversible combination at the substratebinding site. Consequently, experiments were conducted in which each inhibitor was added along with the pyruvate, and 15 min. prior to pyruvate. Results are shown in Fig. 2. In each case simultaneous addition of substrate protected the enzyme from the inhibitor, up to 100% in the case of ketomalonate and p-hydroxyphenylpyruvate, indicating that the inhibitor was bound at the substrate-binding site, a situation compatible with the structures of the inhibitors. Furthermore, there appeared to be no inhibition becoming evident in those vessels in which substrate and inhibitor were added simultaneously, as manifest by the strict linearity of the plots of log (a - x) against time ; that is, the presence of substrate prevented any apparent progressive inhibition. MINUTES Dixon and Webb (2) state that a reversibly FIG. 2. The course of decarboxylation of pyrucombining substrate can never prevent the vate by pyruvic carboxylase with ( l ) no inhibitor ultimate complete inhibition by an irreverspresent, (X) inhibitor added 15 min. before pyible inhibitor, so long as sufficient inhibitor ruvate, and (0) inhibitor added simultaneously is present to saturate the enzyme, since with pyruvate. Inhibitors, with final molar concenthere is always a small amount of free entrations: A, phenylpyruvate, 4.5 X lo-*; B, ketozyme as a result of the reversible equilibmalonate, 9.0 X lo-‘; C, o-nitrophenylpyruvate, rium with the substrate. This free enzyme 4.5 X 10-5; D, p-hydroxyphenylpyruvate, 4.5 X 10m4; is thus subject to the effects of the inhibiE, glyoxylate, 3.2 X 10T6. Pyruvate concentration, 4.5 X lo-’ M. The lines are least-squares lines. tor. The above mentioned linearity of the
ENZYME
KINETICS.
plots of log (a - 5) against time is not necessarily a contradiction of this statement; rather, the period of measurement and experimental error of the method COUplod with the relative affinities of the enzyme for each of the compounds likely rcsulted in inability to detect the progressive inhibition. Since irreversible inhibition is generally characterized by a progressive increase with time, it was considered of interest to determinc the degree of inhibition produced by incubating the enzyme with inhibitor for varying periods of time before addition of substrate. Double side-arm vessels were set up so that each inhibitor could be added t,o the enzyme at precise times from one side arm before addition of substrate from the other. Figure 3 shows the results expressed as logarithm of velocity (= logarithm of enzyme remaining) after preincubation for specific intervals. A logarithmic plot is used t,o show the similarity to a first-order reaction, Thermal inactivation of the enzyme was not a factor in these experiments since all vessels were incubated for an equal time at 30°C. regardless of the interval between addition of inhibit,or and subst,ratc. To determine whether any of these five inhibitors was itself decarboxylated, 2.0 +olcs (45 ~1.) was added from the side arms of vessels containing three times the usual enzyme concentration. At the end of 2 hr. CO2 evolved from each was as follows: glyoxylate, 0; o-nitrophenylpyruvate, 0; kctomalonatc, 0; p-hydroxyphcnylpyruvate, 37 ,J.; phenylpyruvatc, 25 PI. A plot of log ( 0 - .r) against time for the two which lvere decarboxylatetl showed a decreasing velocity with t’imc. Inhibit,ion kinetics of the fire analogs so studied bear :I striking similarit’y to the kinetics of inhibition of specific acetylcholine&erase by certain organic phosphates ( I). Even though thesc latter compounds do not appear to be structural analogs in the general scns:c~,the mechanism of their inhibition of certain estcrases is actually a first-stage hydrolysis, the resulting phosphorylatetl enzyme being quite inactive (2). Xltlridge (1)) working with p-nitrophenyl tlic%hyl thiophosphatc, p-nitrophenyl rii-
0
239
II
5
IO
0 5 MINUTES
IO
0
5
IO
FIG. 3. Progressive decreasein velocity of pyruvic carboxylase by structural analogs of py-
ruvnts when inhibitors were added at certain intervals prior to addition of substrate. Inhibitors, with final molar concentrations: A, phenylpyruvate, 9.0 X 10e4; B, ketomalonate, 9.0 X 10A5; c, o-nitrophenylpyruvate, 4.5 X 10e5; D, p-hydroxyphcnylpgruvate, 4.5 X lo-‘; E, glyoxylate, 1.8 X lOmE.Pyruuate concentration, 4.5 X 10e3 M. The arrows indicate velocity in absence of inhibitor. The lines are least-squares lines.
ethyl phosphate, and S-quinolyl diethyl thiophosphate, found that when either of the first. two of these compounds was added to his cholincsterase preparations at certain times before addition of substrate, a plot of logarithm of percentage activity (= logarithm of velocity) against time of incubation yieldetl a straight line which intercepted the origin. When the quinolyl derivative was used, a straight line again resulted which! howcvcr, did not intercept the origin. Such results wit’h this latter compound led him to deduce that the difference between logarithm 100% activity and the actual intcrccpt of the experimental plot w-as :I measure of reversible inhibition; the progressirc linear portion of the plot was the irreversible portion. ,4n analogous situation appears evident with carboxylase inhibitors. Phenylpyruvatc, o-nitrophenylpyrurate, and glgoxylate each yielded a progressive inhibition which did not intercept the origin. Thr former ~v-ns the only inhibitor the effects of which were partly rcverscd by dialysis. Clpoxylate, phcnyl-
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pyruvate, and its o-nitro derivative thus appeared to yield kinetic results similar to the 8-quinolyl derivative of Aldridge (1) while the other two were more analogous to his p-nitrophenyl derivatives. On the basis of the experimental data presented, an exact mechanism of inhibition cannot be inferred. The evidence indicates t’hat the five analogs studied in most detail combine with the active site of t,he enzyme. The progressive inhibition occurring before addition of substrate could be the result of simply a slow uptake of the inhibitor, the final product being a complex between the enzyme and unaltered analog. Since, however, decarboxylation of two of these apparently occurred, the true inhibitor may have been the aldehyde corresponding to each of the analogs. The fact that decarboxylation of ketomalonate, glyoxylate, or o-nitrophenylpyruvate could not be demonstrated does not necessarily invalidate such a proposal since the aldehydes theoretically thus formed were perhaps more potent inhibitors than the others and the total amount of COZ evolved before complete inactivation of the enzyme could well have
escaped detection due to limitations of the method. The likelihood that a nondissociable bonding occurs between any such aldehydc formed and the enzyme as with inhibition of choline&erase by the organic phosphates seems somewhat, remote. If this were the cast, at any given enzyme concentration the dcgrec of inactivation of the enzyme would be the same upon decarboxylation of a certain number of molecules of any of the inhibitors. The fact that p-hydroxyphenylpyruvate was almost completely decarboxylated at the concentration employed, the velocity decreasing with time, suggests rather that each molecule of aldehyde, if formed, partially inactivates the active site at which it was formed. REFERENCES 1. 2.
J. 46, 451 (1950). C., “Enzymes.” ,4cademic Press, New York, 1958. 3. GREEN, D. E., HERBERT, D., .~SD SUBRAHMANYAN, V., J. Bid. Chem. 138,327 (1941). 4. REDEMANK, C. T., AND MEIKLE, R. W., Arch. Biochem. Biophys. 59, 106 (1955). 5. WATT, D. D., ASD WERKMAN, C. H., Arch. Biothem. Biophys. 50,64 (1954). ALDRIDGE, W. K., Biochem. DIXON, M., AND WEBB, E.