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Kinetic mechanism of guinea-pig skeletal muscle lactate dehydrogenase (M4) with oxaloacetate-NADH and pyruvate-NADH as substrates
Inr J. Brochrm. Vol. 13. pp. 727 to 731. 1981 Printed I” Great Britatn. All rights reserved
0020-71 IXl81/060727-05SOZ.O9/0 CopyrIght 0 1981 Pergamon Press Ltd
KINETIC MECHANISM OF GUINEA-PIG SKELETAL MUSCLE LACTATE DEHYDROGENASE (M4) WITH OXALOACETATE-NADH AND PYRUVATE-NADH AS SUBSTRATES S. Department
of Biochemistry,
SEMPERE,
Faculty
A.
and J. BOZAL
CORT~
of Chemistry,
University
of Barcelona
(Central),
Spain
(Receiced 24 Noaember 1980) Abstract-l. Guinea-pig skeletal muscle lactate dehydrogenase M4 isoenzyme, with pyruvate and NADH as substrates, is adapted to an ordered bi-bi ternary complex mechanism at pH 7.0. 2. In the same conditions, the kinetic mechanism of the reaction, with oxaloacetate and NADH as substrates, is of the rapid ordered bi-bi ternary complex type; NADH is the first substrate . equilibrium . in the reaction sequence.
INTRODUCTION
Knowledge of the protein structures of dogfish lactate dehydrogenase (EC 1.1.1.27) (Rossmann et al., 1967; Adams et al., 1970; Adams et al., 1973). pig cytoplasmic malate dehydrogenase (EC 1.1.1.37) (Hill et al., 1972; Webb et al., 1973), horse alcohol dehydrogenase (EC 1.1.1.1) (Branden et al.. 1973; Eklund et al., 1974) and lobster glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) (Buehner et al., 1973; Buehner et al., 1974), with a high degree of resolution, shows that the regions of their polypeptide chains to which the NAD+ binds have very similar structures. The main structural elements involved are six strand of parallel sheet and four r-helices (Hill et al., 1972). The analogy of their catalytic domains (Hill et al.. 1972; Rao & Rossmann. 1973) appears in lactate dehydrogenase and in malate dehydrogenase as a main feature, differentiating them from the other two dehydrogenases; both also have an additional x-helix segment in the NAD+ binding region (Hill et al., 1972). The intimate correlation between the structures of pig heart malate dehydrogenase and lactate dehydrogenase has been shown by Hill et al. (1972). The fact that both dehydrogenases are adapted to homologous kinetic mechanisms (Lodola et al., 1978) and the close resemblance of the dimer of cytoplasmic malate dehydrogenase to one-half of the lactate dehydrogenase molecule, led Lodola er al. (1978) to postulate that they are the product of the divergent evolution from a common ancestor. Chicken liver lactate dehydrogenase predominance of H4 (CortCs & Bozal, 1972) and the five guinea-pig skeletal muscle lactate dehydrogenase isoenzymes catalyse the reduction of the oxaloacetate by the NADH (Busquets et al.. 1979; Puig et al., 1980) contrary to what had been postulated previously (Meister, 1950), the activity with L-malate and NAD+ being insignificant. This fact suggests the existance of a binding “locus” common in both dehydrogenases for the oxaloacetate and may support the hypothesis of Lodola et al. (1978). In this paper, there is established for the first time the kinetic mechanism of the guinea-pig skeletal
muscle lactate dehydrogenase M, isoenzyme, which is predominant in the tissue (Olsson, 1975) with oxaloacetate and NADH as substrates. The results obtained are compared with those obtained from performing a similar study with NADH and pyruvate. preferred substrate of the enzyme. MATERIALS
AND METHODS
Reagents Oxaloacetic acid. ot_-malic acid and sodium DL-lactate (Merck); sodium pyruvate, NADH and NAD+ (Boehringer); oxamic acid (Sigma). All other chemicals used were reagent grade. Kinetic studies The initial velocities of enzyme reduction of the pyruvate or of the oxaloacetate by the NADH were determined at 30 + O.l”C and pH 7.0 (50mM phosphate buffer) by measurement of the absorbance changes of the NADH at 340 nm (E = 6.22’ IO3 cm* mol-‘) in a Beckman model 25 recording spectrophotometer, in 3 ml and 1cm light path cells. The reaction being started with 0.7 nkat of M, isoenzyme, free from malate dehydrogenase, purified by the method of Puig et al. (1980) and its specific activity was 4.7 pkat/mg. The kinetic mechanism of the systems being studied was established by the reaction product inhibition method with respect to each substrate. in the presence of a constant concentration, saturating and non-saturating, of the other substrate. It was confirmed, moreover, by the study of oxamate inhibition, which originates characteristic “dead-end” complexes. Substrate inhibition was studied by application of the Dalziel’s (1957) method.
RESULTS AND DISCUSSION Initial oelocites in absence
of products
The optimum pH for the M, isoenzyme with a 0.1 mM pyruvate and 0.08 mM NADH was determined in a 50mM, pH 5.7-8.0, sodium phosphate buffer and was found to be 6.0-6.2, similar to the value described for lactate dehydrogenase from other sources (Fritz, 1967; FernBndez-Santos et al., 1979). A value of 6.2-6.4 was obtained under the same con727
S. SEMPEREet al.
728
r
0.5
10
1.5
21)
10
20
30
[OAA]e’(mM)e’
40
50
[NADH].‘(mM1’
Fig. I. Initial reaction rates of oxaloacetateelactate dehydrogenaseeNADH system. Reactions were carried out in 50mM. pH 7.0 phosphate buffer. (a) [NADH]: I, 0.02 mM; 2. 0.04mM; 3. 0.06mM: and 4. 0.08 mM. (b) [Oxaloacetate]: I. 0.9 mM: 2. I.5 mM: 3. 2 mM: 4, 3 mM: and 5. 4mM.
ditions with 1mM oxaloacetate and 0.08 mM NADH. The enzyme becomes inactivated at the optimum pH with the passage of time, whereby the kinetic studies were performed at pH 7.0 at which level its activity is 80?,, of that exhibited at the optimum pH and remains constant with time. The double reciprocal plot with oxaloacetate and NADH as substrates gives straight lines with a common intercept to the left of the ordinates axis if the NADH is the variable substrate and on the axis if the oxaloacetate is variable (Fig. 1). It is characteristic of a rapid equilibrium ordered bireactant system (Segel, 1975a). Non-parallel straight lines, having a common intercept to the left of the ordinates axis. are always obtained with pyruvate and NADH as substrates
(Fig. 2) suggesting that the general mechanism is of the sequential type (Cleland, 1963). The values of the kinetic parameters of both systems. deduced from the corresponding replots (Florini & Vestling, 1957; Segel, 1975a). are: Kpyruvrle = 0.1 I mM ; K NADH= 0.008 mM; K0X3,0~CUld,P = 0.81 mM and K NADH= 0.07mM. Rractim
product inhibition
With pyruvate and NADH as substrates, the results obtained (Table 1) agree with those of an ordered bi-bi ternary complex mechanism. in which the coenzyme is the first substrate to interact with the enzyme. This mechanism is the one to which lactate dehydrogenase from different sources habitually adapts (Cartes & Bozal. 1973; Holbrook rt al., 1975; Fernan-
0.5 -Ao-o.-*-
(a)
5 5
10
15
20 [Pyr ]-‘(mM)d’
[NADH].‘(mMy’
Fig, 2. Initial reaction rates of pyruvateelactate dehydrogenase-NADH system. (a) [NADH]; 0.02mM: 2. 0.04mM: 3. 0.054mM. and 4. 0.08mM. (b) [Pyruvate]: 0.5mM: 2. 0.1 mM, 3. 0.2mM: 0.3 mM: and 5. 0.6 mM. The conditions used were as described in Fig. I.
1, 4.
Kinetic Table
1. Reaction
product
inhibition
mechanism
of LDH
of pyruvate-lactate Variable
NADH
NAD’
L-Lactate (0.01-0.07
mM)
(C) Competitive:
NC K,, = 0.014 mM
K,, = 0.013 mM (NC) non-competitive:
2. Reaction
product
inhibition
UC
Inhibitor NAD+ (0.1-0.5 mM)
r-Malate (10-50mM)
C Ki, = 0.65 mM
NC
K,, = 1.9mM K,, = 0.82 mM
K,, = 11.4mM K,, = 5.1 mM
NC K,, = 0.017 mM
NC Ki, = 0.018 mM K,, = 0.074 mM
& = 0.052 mM
K,, = 0.03 mM (UC) uncompetitive.
petitive inhibition of the L-malate vs the NADH (saturating oxaloacetate) and in the non-competitive inhibition of the NAD+ vs the oxaloacetate (saturating NADH). According to the theoretical predictions (Segel, 1975b) there should be no inhibition in either case; nevertheless. since the inhibition exercised by the L-malate and the oxaloacetate or the NAD+ and the NADH between them is competitive (Table 2). the saturating concentrations determined in the absence of products are no longer saturating under these conditions and it would be necessary to increase them considerably to cancel out the inhibition. This is in agreement with the increase in the inhibition constants on raising the concentration of the constant substrate (Table 2. Segel. 1975b). The mechanism formulated for the M, isoenzyme with oxaloacetate and NADH as substrates is analogous to the one deduced on studying the reduction of hydroxypyruvate with NADH, catalysed by chicken liver lactate dehydrogenase (Lluis & Bozal, 1978). Substrate
inhibition
Pyruvate or oxaloacetate concentrations above 0.8 and 4 mM, respectively, inhibit the ML isoenzyme by excess substrate. In both cases, the inhibition is uncompetitive and linear vs the NADH and, according to the mechanisms postulated, it is due to the formation of the E-NAD+-pyruvate and EFNAD+oxaloacetate abortive complexes.
of oxaloacetate-lactate pH 7.0.
(0.015-0.08
Oxaloacetate non-saturating 1mM
NADH saturating 0.08 mM
NC
Variable NADH
(0.1-0.8 mM)
NADH non-saturating 0.02 mM
K,, = 0.8 mM
dez-Santos et a/.. 1979). The only difference between the theoretical predictions (Cleland, 1963) and the results of the experiments is to be found in that when the pyruvate is the variable substrate and the NAD+ is the inhibiting product, the inhibition should be cancelled out in presence of a saturating concentration of NADH. The linear non-competitive inhibition described here shows that since the NAD+ and the NADH are competing for the same enzyme form, the kinetically saturating concentration of NADH (0.08 mM), deduced in absence of the reaction products, ceases to be saturating in the presence of NAD+, whereby the inhibition subsists. Nevertheless, the percentage inhibition decreases, since the values of K, and Kii are higher than those obtained when using a lower constant NADH concentration (0.02 mM) (Table 1). This is an evidence that the concentrations of NAD+ tested would not cause inhibition if the NADH concentration could be considerably increased (Segel. 1975b). When using oxaloacetate and NADH as substrates, the inhibition by the reaction products (Table 2) is adapted basically to that described for a rapid equilibrium ordered bi-bi ternary complex mechanism, with the existence of active enzyme-coenzyme binary complexes and of the abortive E-NAD+-oxaloacetate and E-NADH-L-malate complexes, the limiting step of the velocity being the release of the L-malate (Segel, 1975b). The only discrepancies appear in the uncom-
Table
Pyruvate
C
Ki, = 0.75 mM
system at pH 7.0.
substrate
Pyruvate saturating 0.8 mM
C
(0.3-3 mM)
dehydrogenase-NADH
(0.015~0.08 mM)
Pyruvate non-saturating 0.2 mM
Inhibitor
729
mM)
Oxaloacetate saturating 4mM C K,, = 0.82 mM
UC
UC
Ki, = 15mM
K,, = 67.5mM
dehydrogenase-NADH
system
substrate Oxaloacetate NADH non-saturating 0.02 mM NC
(0.84
mM)
NADH saturating 0.08 mM NC
K,, = 0.34mM Kii = 1.37 mM
K,, = 1.8mM Ki, = 5.82 mM
C K,, = 19 mM
C K,, = 21 mM
at
S. SEMPERE et al.
730
,,5
lJi ”
rmol
NADH j’ min
0.6.
0.4
(b) “L
30
10
[NADH].‘(mM1’ Fig. 3. Inhibition
of the pyruvate-lactate
dehydrogenase-NADH
and oxaloacetate-lactate
dehydroge-
nase-NADH systems by oxamate. (a) [Pyruvate] = 0.2 mM; (b) [Oxaloacetate] = 1 mM. (a) [Oxamate] : 1. 0.06mM; 2, 0.03mM: 3, 0.015mM; and 4, OmM. (b) [Oxamate]: I. 0.08mM; 2. 0.06mM: 3. 0.03 mM; and 4, 0 mM. The conditions Oxamate
inhibition
used were as described
in Fig.
I,
REFERENCES
The oxamate is a competitive inhibitor of lactate dehydrogenase from various sources vs the pyruvate (Nisselbaum et al., 1964). The different mechanisms deduced for the systems under study make their use advisable, since the type of inhibition it should have vs the NADH should be identical, irrespectively as to whether the pyruvate or the oxaloacetate is used as the constant substrate. The results of the experiments agree with the theoretical predictions and show that in both systems the oxamate inhibition vs the NADH is linearly uncompetitive (Fig. 3), the values of the inhibition constants being 0.094mM (0.2 mM pyruvate) and 0.13 mM (1 mM oxaloacetate). This inhibition is justified, according to the mechanisms postulated, by the formation of the abortive E-NADHoxamate complex. This means a competition of the inhibitor for the pyruvate and the oxaloacetate binding site, in agreement with the fact that the oxamate competitively inhibits guinea-pig skeletal muscle lactate dehydrogenase M, isoenzyme vs pyruvate and oxaloacetate (Puig et al., 1980).
SUMMARY
The application of the reaction product inhibition method shows that at pH 7.0, the reduction of the oxaloacetate. catalysed by the guinea-pig skeletal muscle lactate dehydrogenase M, isoenzyme, is adapted to a rapid equilibrium ordered bi-bi ternary complex mechanism, for which there is postulated the existence of the abortive E-NAD+-oxaloacetate and E-NADH-L-malate complexes. The mechanism differs from the one deduced under the same conditions with pyruvate and NADH, which is ordered bi-bi ternary complex with the abortive E-NAD+pyruvate complex. As oxamate acts as inhibitor of both systems, it produces the abortive E-NADHoxamate complex, which confirms the proposed mechanisms.
ADAMS M. J., FORD G. C., KOEKOEK R.. LENTZ P. J., MCPHERSON A. JR, ROSSMANN M. G.. SMILEY 1. E.. SCHENTZ R. W. & WONACOTT A. J. (1970) Structure of lactate dehydrogenase at 2.8 A resolution. Nature 227, 1098-l 103. ADAMS M. J., BUEHNER M.. CHANDRASEKHAR K.. FORD G. C.. HACKERT M. L.. LILJAS A., RO~~MANN M. G., SMILEY I. E., ALLISON W. S., EVERSE J., KAPLAN N. 0. & TAYLOR S. S. (1973) Structure-function relations in lactate dehydrogenase. Proc. natn. Acad. Sci. U.S.A. 70, 1968-1972. BRANDEN C. I., EKL~ND H., NORDSTROM B.. Bo~wt T.. SODERLUND G.. ZEPPEZAUER E., OHLSSON 1. & AKESON A. (1973) Structure of liver alcohol dehydrogenase at 2.9A resolution. Proc. narn. Acad. Sci. U.S.A. 70. 2439-2442. BUEHNER M.. FORD G. C.. MORAS D., OLSEN K. W. & ROSSMANN M. G. (1973) o-Glyceraldehyde-3-phosphate dehydrogenase. Three-dimensional structure and evolutionary significance. Proc. natn. Acad. Sci. U.S.A. 70, 3052-3054. BUEHNER M.. FORD G. C.. MORAS D.. OLSEN K. W. & ROSSMANN M. G. (1974) Three-dimensional structure of o-glyceraldehyde 3-phosphate dehydrogenase. J. molec. Biol. 90, 25-49. BUSQUETSM., BARO J., COR~ES A. & BOZAL J. (1979) Separation and properties of the two forms of chicken liver cytoplasmic malate dehydrogenase. Inr. J. Biochem. IO. 823-835. CLELAND W. W. (1963) The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochirn. hiophys. Actu 67, 104137. CORTES A. & BOZAL J. (1972) Kinetic studies with lactic dehydrogenase. Recta esp. Fisiol. 28. 297-308. CORTES A. & BOZAL J. (1973) Reaction mechanism of lactate dehydrogenase. Rerta es-p. Fisiol. 29, 131-144. DALZIEL K. (1957) Initial steady-state velocities in the reaction evaluation of enzymeecoenzymeesubstrate mechanisms. Acta them. wand. 11, 170& 1723. EKLUND H.. NORDSTROM B.. ZEPPEZAUER E., SO~ERLUND G.. OHLSSON 1.. BOIWE T. & BRANDEN C. I. (1974) Structure of horse liver alcohol dehydrogenase. FEB.5 Left. 44, 200-204.
Kinetic mechanism of LDH
731
FERNANDEZ-SANTOS T.. LLUIS C. & BOZALJ. (1979) Aborheart lactic dehydrogenase variants on nucleotide anative complexes as a cause of substrate inhibition of rablogues and on substrate analogues in the absence and in bit muscle lactate dehydrogenase. J. m&c. Catalysis 5. the presence of oxalate and oxamate. J. hiol. Chem. 239. 247-262. 283&2834. FLORINIJ. R. & VESTLINGC. S. (1957) Graphical determiOL~SON S. 0. R. (1975) Comparative studies on the temnation of the dissociation constants for two-substrate perature dependence of lactic and malid dehydrogenase enzyme systems. Biochim. biophys. Acra 25, 575-578. from a homeotherm, guinea-pig; two hibernators. hedgeFRITZ P. J. (1967) Rabbit lactate dehydrogenase isozymes: hog and bat; and two poikilotherms. frog and cod. effect of pH on activity. Science 156. 82-83. Comp. Biochem. Physiol. 15, 5-18. HILL E. J.. TSERN~GLOUD.. WEBBL. E. & BANASZAKL. J. PUIG J.. CORPUSA. & BOZALJ. (1980) Influence of lactate dehydrogenase on the kinetic and electrophoretic behav(1972) Polypeptide conformation of cytoplasmic malate iour of guinea-pig skeletal muscle cvtoplasmic malate dehydrogenase from an electron density map at 3.0A resolution. J. molec. Biol. 72. 577-591. dehydrogenase. Iit. J. Biochem. 11. 2i9-i36. HOLBR~OKJ. J., LILJAS A., STEINDELS. J. & ROSSMANN RAO S. T. & ROSSMANNM. G. (1973) Comoarison of suoersecondary structures in pioteins. J. ‘molrc. Biol. ‘76, M. G. (1975) Lactate dehydrogenase. The Enzymes 11, 191-292. 24 l-256. LLUISC. & BOZALJ. (1978) Kinetic mechanism of the hyROSSMANN M. G., JEFFERYB. A.. MAIN P. & WARRENS. droxypyruvate-lactate dehydrogenase-NADH system. (1967) The crystal structure of lactic dehydrogenase. Biochim.
biophgs.
Acta 523. 273-282.
LODOLAA., SHOREJ. D.. PARKERD. M. & HOLBR~OKJ. J. (1978) Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase. Biochem. J. 175, 987-998.
MEISTERA. (1950) Reduction of a. y-diketo and a-keto acids catalyzed by muscle preparations and by crystalline lactic dehydrogenase. J. biol. Chem. 184, I 17-l 29. NISSELBAUM J. S., PACKER D. E. & BODANSKY0. (1964) Comparison of the actions of human brain. liver and
Proc. natn. Acad. Sci. U.S.A. 57. 515-524. Behavior arid of Rapid Equilibrium and Steady-Starr En-_ymr pp. 273-345. Wiley. New York. SEGELI. H. (I 975b) Enzyme kinetics. Behavior aud of Rapid Equilibrium and Steady-Starr En-_yme pp. 505-845. Wiley, New York.
SEGELI. H. (1975a) Enzyme kinetics.
Analysis Sysrems. Analysis Systems.
WEBBL. E., HILL E. J. & BANASZA~L. J. (1973) Conformation of nicotinamide adenine dinucleotide bound to cytoplasmic malate dehydrogenase. Biochemistry 12. 5101~5lG9
0020-71 IXl81/060727-05SOZ.O9/0 CopyrIght 0 1981 Pergamon Press Ltd
KINETIC MECHANISM OF GUINEA-PIG SKELETAL MUSCLE LACTATE DEHYDROGENASE (M4) WITH OXALOACETATE-NADH AND PYRUVATE-NADH AS SUBSTRATES S. Department
of Biochemistry,
SEMPERE,
Faculty
A.
and J. BOZAL
CORT~
of Chemistry,
University
of Barcelona
(Central),
Spain
(Receiced 24 Noaember 1980) Abstract-l. Guinea-pig skeletal muscle lactate dehydrogenase M4 isoenzyme, with pyruvate and NADH as substrates, is adapted to an ordered bi-bi ternary complex mechanism at pH 7.0. 2. In the same conditions, the kinetic mechanism of the reaction, with oxaloacetate and NADH as substrates, is of the rapid ordered bi-bi ternary complex type; NADH is the first substrate . equilibrium . in the reaction sequence.
INTRODUCTION
Knowledge of the protein structures of dogfish lactate dehydrogenase (EC 1.1.1.27) (Rossmann et al., 1967; Adams et al., 1970; Adams et al., 1973). pig cytoplasmic malate dehydrogenase (EC 1.1.1.37) (Hill et al., 1972; Webb et al., 1973), horse alcohol dehydrogenase (EC 1.1.1.1) (Branden et al.. 1973; Eklund et al., 1974) and lobster glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) (Buehner et al., 1973; Buehner et al., 1974), with a high degree of resolution, shows that the regions of their polypeptide chains to which the NAD+ binds have very similar structures. The main structural elements involved are six strand of parallel sheet and four r-helices (Hill et al., 1972). The analogy of their catalytic domains (Hill et al.. 1972; Rao & Rossmann. 1973) appears in lactate dehydrogenase and in malate dehydrogenase as a main feature, differentiating them from the other two dehydrogenases; both also have an additional x-helix segment in the NAD+ binding region (Hill et al., 1972). The intimate correlation between the structures of pig heart malate dehydrogenase and lactate dehydrogenase has been shown by Hill et al. (1972). The fact that both dehydrogenases are adapted to homologous kinetic mechanisms (Lodola et al., 1978) and the close resemblance of the dimer of cytoplasmic malate dehydrogenase to one-half of the lactate dehydrogenase molecule, led Lodola er al. (1978) to postulate that they are the product of the divergent evolution from a common ancestor. Chicken liver lactate dehydrogenase predominance of H4 (CortCs & Bozal, 1972) and the five guinea-pig skeletal muscle lactate dehydrogenase isoenzymes catalyse the reduction of the oxaloacetate by the NADH (Busquets et al.. 1979; Puig et al., 1980) contrary to what had been postulated previously (Meister, 1950), the activity with L-malate and NAD+ being insignificant. This fact suggests the existance of a binding “locus” common in both dehydrogenases for the oxaloacetate and may support the hypothesis of Lodola et al. (1978). In this paper, there is established for the first time the kinetic mechanism of the guinea-pig skeletal
muscle lactate dehydrogenase M, isoenzyme, which is predominant in the tissue (Olsson, 1975) with oxaloacetate and NADH as substrates. The results obtained are compared with those obtained from performing a similar study with NADH and pyruvate. preferred substrate of the enzyme. MATERIALS
AND METHODS
Reagents Oxaloacetic acid. ot_-malic acid and sodium DL-lactate (Merck); sodium pyruvate, NADH and NAD+ (Boehringer); oxamic acid (Sigma). All other chemicals used were reagent grade. Kinetic studies The initial velocities of enzyme reduction of the pyruvate or of the oxaloacetate by the NADH were determined at 30 + O.l”C and pH 7.0 (50mM phosphate buffer) by measurement of the absorbance changes of the NADH at 340 nm (E = 6.22’ IO3 cm* mol-‘) in a Beckman model 25 recording spectrophotometer, in 3 ml and 1cm light path cells. The reaction being started with 0.7 nkat of M, isoenzyme, free from malate dehydrogenase, purified by the method of Puig et al. (1980) and its specific activity was 4.7 pkat/mg. The kinetic mechanism of the systems being studied was established by the reaction product inhibition method with respect to each substrate. in the presence of a constant concentration, saturating and non-saturating, of the other substrate. It was confirmed, moreover, by the study of oxamate inhibition, which originates characteristic “dead-end” complexes. Substrate inhibition was studied by application of the Dalziel’s (1957) method.
RESULTS AND DISCUSSION Initial oelocites in absence
of products
The optimum pH for the M, isoenzyme with a 0.1 mM pyruvate and 0.08 mM NADH was determined in a 50mM, pH 5.7-8.0, sodium phosphate buffer and was found to be 6.0-6.2, similar to the value described for lactate dehydrogenase from other sources (Fritz, 1967; FernBndez-Santos et al., 1979). A value of 6.2-6.4 was obtained under the same con727
S. SEMPEREet al.
728
r
0.5
10
1.5
21)
10
20
30
[OAA]e’(mM)e’
40
50
[NADH].‘(mM1’
Fig. I. Initial reaction rates of oxaloacetateelactate dehydrogenaseeNADH system. Reactions were carried out in 50mM. pH 7.0 phosphate buffer. (a) [NADH]: I, 0.02 mM; 2. 0.04mM; 3. 0.06mM: and 4. 0.08 mM. (b) [Oxaloacetate]: I. 0.9 mM: 2. I.5 mM: 3. 2 mM: 4, 3 mM: and 5. 4mM.
ditions with 1mM oxaloacetate and 0.08 mM NADH. The enzyme becomes inactivated at the optimum pH with the passage of time, whereby the kinetic studies were performed at pH 7.0 at which level its activity is 80?,, of that exhibited at the optimum pH and remains constant with time. The double reciprocal plot with oxaloacetate and NADH as substrates gives straight lines with a common intercept to the left of the ordinates axis if the NADH is the variable substrate and on the axis if the oxaloacetate is variable (Fig. 1). It is characteristic of a rapid equilibrium ordered bireactant system (Segel, 1975a). Non-parallel straight lines, having a common intercept to the left of the ordinates axis. are always obtained with pyruvate and NADH as substrates
(Fig. 2) suggesting that the general mechanism is of the sequential type (Cleland, 1963). The values of the kinetic parameters of both systems. deduced from the corresponding replots (Florini & Vestling, 1957; Segel, 1975a). are: Kpyruvrle = 0.1 I mM ; K NADH= 0.008 mM; K0X3,0~CUld,P = 0.81 mM and K NADH= 0.07mM. Rractim
product inhibition
With pyruvate and NADH as substrates, the results obtained (Table 1) agree with those of an ordered bi-bi ternary complex mechanism. in which the coenzyme is the first substrate to interact with the enzyme. This mechanism is the one to which lactate dehydrogenase from different sources habitually adapts (Cartes & Bozal. 1973; Holbrook rt al., 1975; Fernan-
0.5 -Ao-o.-*-
(a)
5 5
10
15
20 [Pyr ]-‘(mM)d’
[NADH].‘(mMy’
Fig, 2. Initial reaction rates of pyruvateelactate dehydrogenase-NADH system. (a) [NADH]; 0.02mM: 2. 0.04mM: 3. 0.054mM. and 4. 0.08mM. (b) [Pyruvate]: 0.5mM: 2. 0.1 mM, 3. 0.2mM: 0.3 mM: and 5. 0.6 mM. The conditions used were as described in Fig. I.
1, 4.
Kinetic Table
1. Reaction
product
inhibition
mechanism
of LDH
of pyruvate-lactate Variable
NADH
NAD’
L-Lactate (0.01-0.07
mM)
(C) Competitive:
NC K,, = 0.014 mM
K,, = 0.013 mM (NC) non-competitive:
2. Reaction
product
inhibition
UC
Inhibitor NAD+ (0.1-0.5 mM)
r-Malate (10-50mM)
C Ki, = 0.65 mM
NC
K,, = 1.9mM K,, = 0.82 mM
K,, = 11.4mM K,, = 5.1 mM
NC K,, = 0.017 mM
NC Ki, = 0.018 mM K,, = 0.074 mM
& = 0.052 mM
K,, = 0.03 mM (UC) uncompetitive.
petitive inhibition of the L-malate vs the NADH (saturating oxaloacetate) and in the non-competitive inhibition of the NAD+ vs the oxaloacetate (saturating NADH). According to the theoretical predictions (Segel, 1975b) there should be no inhibition in either case; nevertheless. since the inhibition exercised by the L-malate and the oxaloacetate or the NAD+ and the NADH between them is competitive (Table 2). the saturating concentrations determined in the absence of products are no longer saturating under these conditions and it would be necessary to increase them considerably to cancel out the inhibition. This is in agreement with the increase in the inhibition constants on raising the concentration of the constant substrate (Table 2. Segel. 1975b). The mechanism formulated for the M, isoenzyme with oxaloacetate and NADH as substrates is analogous to the one deduced on studying the reduction of hydroxypyruvate with NADH, catalysed by chicken liver lactate dehydrogenase (Lluis & Bozal, 1978). Substrate
inhibition
Pyruvate or oxaloacetate concentrations above 0.8 and 4 mM, respectively, inhibit the ML isoenzyme by excess substrate. In both cases, the inhibition is uncompetitive and linear vs the NADH and, according to the mechanisms postulated, it is due to the formation of the E-NAD+-pyruvate and EFNAD+oxaloacetate abortive complexes.
of oxaloacetate-lactate pH 7.0.
(0.015-0.08
Oxaloacetate non-saturating 1mM
NADH saturating 0.08 mM
NC
Variable NADH
(0.1-0.8 mM)
NADH non-saturating 0.02 mM
K,, = 0.8 mM
dez-Santos et a/.. 1979). The only difference between the theoretical predictions (Cleland, 1963) and the results of the experiments is to be found in that when the pyruvate is the variable substrate and the NAD+ is the inhibiting product, the inhibition should be cancelled out in presence of a saturating concentration of NADH. The linear non-competitive inhibition described here shows that since the NAD+ and the NADH are competing for the same enzyme form, the kinetically saturating concentration of NADH (0.08 mM), deduced in absence of the reaction products, ceases to be saturating in the presence of NAD+, whereby the inhibition subsists. Nevertheless, the percentage inhibition decreases, since the values of K, and Kii are higher than those obtained when using a lower constant NADH concentration (0.02 mM) (Table 1). This is an evidence that the concentrations of NAD+ tested would not cause inhibition if the NADH concentration could be considerably increased (Segel. 1975b). When using oxaloacetate and NADH as substrates, the inhibition by the reaction products (Table 2) is adapted basically to that described for a rapid equilibrium ordered bi-bi ternary complex mechanism, with the existence of active enzyme-coenzyme binary complexes and of the abortive E-NAD+-oxaloacetate and E-NADH-L-malate complexes, the limiting step of the velocity being the release of the L-malate (Segel, 1975b). The only discrepancies appear in the uncom-
Table
Pyruvate
C
Ki, = 0.75 mM
system at pH 7.0.
substrate
Pyruvate saturating 0.8 mM
C
(0.3-3 mM)
dehydrogenase-NADH
(0.015~0.08 mM)
Pyruvate non-saturating 0.2 mM
Inhibitor
729
mM)
Oxaloacetate saturating 4mM C K,, = 0.82 mM
UC
UC
Ki, = 15mM
K,, = 67.5mM
dehydrogenase-NADH
system
substrate Oxaloacetate NADH non-saturating 0.02 mM NC
(0.84
mM)
NADH saturating 0.08 mM NC
K,, = 0.34mM Kii = 1.37 mM
K,, = 1.8mM Ki, = 5.82 mM
C K,, = 19 mM
C K,, = 21 mM
at
S. SEMPERE et al.
730
,,5
lJi ”
rmol
NADH j’ min
0.6.
0.4
(b) “L
30
10
[NADH].‘(mM1’ Fig. 3. Inhibition
of the pyruvate-lactate
dehydrogenase-NADH
and oxaloacetate-lactate
dehydroge-
nase-NADH systems by oxamate. (a) [Pyruvate] = 0.2 mM; (b) [Oxaloacetate] = 1 mM. (a) [Oxamate] : 1. 0.06mM; 2, 0.03mM: 3, 0.015mM; and 4, OmM. (b) [Oxamate]: I. 0.08mM; 2. 0.06mM: 3. 0.03 mM; and 4, 0 mM. The conditions Oxamate
inhibition
used were as described
in Fig.
I,
REFERENCES
The oxamate is a competitive inhibitor of lactate dehydrogenase from various sources vs the pyruvate (Nisselbaum et al., 1964). The different mechanisms deduced for the systems under study make their use advisable, since the type of inhibition it should have vs the NADH should be identical, irrespectively as to whether the pyruvate or the oxaloacetate is used as the constant substrate. The results of the experiments agree with the theoretical predictions and show that in both systems the oxamate inhibition vs the NADH is linearly uncompetitive (Fig. 3), the values of the inhibition constants being 0.094mM (0.2 mM pyruvate) and 0.13 mM (1 mM oxaloacetate). This inhibition is justified, according to the mechanisms postulated, by the formation of the abortive E-NADHoxamate complex. This means a competition of the inhibitor for the pyruvate and the oxaloacetate binding site, in agreement with the fact that the oxamate competitively inhibits guinea-pig skeletal muscle lactate dehydrogenase M, isoenzyme vs pyruvate and oxaloacetate (Puig et al., 1980).
SUMMARY
The application of the reaction product inhibition method shows that at pH 7.0, the reduction of the oxaloacetate. catalysed by the guinea-pig skeletal muscle lactate dehydrogenase M, isoenzyme, is adapted to a rapid equilibrium ordered bi-bi ternary complex mechanism, for which there is postulated the existence of the abortive E-NAD+-oxaloacetate and E-NADH-L-malate complexes. The mechanism differs from the one deduced under the same conditions with pyruvate and NADH, which is ordered bi-bi ternary complex with the abortive E-NAD+pyruvate complex. As oxamate acts as inhibitor of both systems, it produces the abortive E-NADHoxamate complex, which confirms the proposed mechanisms.
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