Inhibition of Human Erythrocyte Lactate Dehydrogenase by High Concentrations of Pyruvate

Transcription

1 and Eur. J. Biochem. 78, (1977) Inhibition of Human Erythrocyte Lactate Dehydrogenase by High Concentrations of Pyruvate Evidence for the Competitive Substrate Inhibition Chi-Sun WANG Lipoprotein Laboratory, Oklahoma Medical Research Foundation, Oklahoma City (Received December 21, 1976/April 6, 1977) The mechanism of the inhibitory effect of high concentrations of pyruvate on human erythrocyte lactate dehydrogenase has been studied by the use of a new parameter, A, defined as the difference between the reciprocals of initial reaction rates obtained from experimental measurements and hypothetical linear Lineweaver-Burk plots. This parameter served as a method for differentiating between the competitive and uncompetitive substrate inhibition. Results of this study indicate that pyruvate is a competitive substrate inhibitor. It is suggested that the inhibitory effect of pyruvate is due to its competition with NADH for binding to the free enzyme and formation of an inactive enzyme. pyruvate binary complex. The competitive nature of pyruvate inhibition is further supported by the results of a kinetic study with NADH as the variable substrate. The dissociation constant of the inactive enzyme. pyruvate binary complex was determined to be 101 pm. The physiological significance of the inhibitory effect could be to preserve a level of NADH concentration necessary for other vital enzymic reactions of living cells despite the presence of a high concentration of pyruvate. Two mechanisms have been proposed to explain the inhibitory effect exhibited by high concentrations of pyruvate on lactate dehydrogenase activity. One of these is based on the formation of an abortive ternary complex involving enzyme, + NAD pyruvate [l- 51. The other has been attributed to the presence in the pyruvate preparations of a contaminating inhibitor or tautomeric isomer end-pyruvate [6,7]. I have observed in the studies of the human erythrocyte glycolysis that the inhibitory effect of pyruvate on lactate dehydrogenase depends on the concentration of NADH cofactor. The inhibitory effect of pyruvate is compatible with the competitive substrate inhibition mechanism as theoretically predicted by Dalziel [S]. Such an inhibitory effect is a specific phenomenon for the ordered multiple-substrate system when a substrate, other than the first substrate, binds to the free enzyme and forms an inactive binary complex. Therefore, in contrast to the proposal that an abortive complex formation or a contaminating inhibitor of the substrate preparation is responsible for inhibition, results of this kinetic Enzymes. Lactate dehydrogenase (EC ); glyceraldehyde- 3-phosphate dehydrogenase (EC ) ; glucose-6-phosphate dehydrogenase (EC ) ; aldolase (EC ) ; 3-phosphoglycerate kinase (EC ). study suggest that the enzyme. pyruvate binary complex formation is responsible for the inhibition of lactate dehydrogenase at high concentrations of pyruvate. EXPERIMENTAL PROCEDURES Materials The partially purified human erythrocyte lactate dehydrogenase, which had a specific activity of 400 U/mg, and other chemicals were obtained from Sigma Chemical Company. The sodium dodecyl sulfate/polyacrylamide gel electrophoresis [9] pattern indicated that the enzyme preparation contains a major subunit polypeptide band with a molecular weight of and a minor protein band of Enzymic assays [lo] indicated that the commercial preparation of lactate dehydrogenase is free from other glycolytic enzymes such as glyceraldehyde- 3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, aldolase and 3-phosphoglycerate kinase. Preparative isoelectric focusing indicated that the commercial preparation consisted mainly of the LDH- 1 (H4) isozyme activity [Ill with an isoelectric point of 4.4. The LDH-2 (H3M) activity, pl 5.1, was only

2 1 570 Competitive Substrate Inhibition present in trace amounts ; therefore, further purification of the lactate dehydrogenase preparation was not attempted. Spectrophotometric Measurement The rates of oxidation of NADH and reduction of NAD were determined in a Beckman model 25 spectrophotometer with a recorder and temperature control unit attached. The enzyme reactions were performed at 37 C in the presence of 50 mm Tris- HC1, ph 7.4, in a final volume of 3ml. Initial reaction rates were determined from the linear portion of the absorbance change at 340 nm. A full scale sensitivity of absorbance unit and a chart speed of 1 inimin (2.5 cm/min) were used. The unit of activity (U) was defined as the amount of enzyme which catalyzed the formation of 1 pmol of product per min l/pyruvate (mm- ) Analysis of Results The linearity of all plots was checked graphically and the data were analyzed in a Compucorp 344 Micro Statistician computer. The data were analyzed by linear regression and the coefficients of variation (c.v.) were within 2 of high substrate concentrations and 10 of the low substrate concentrations. Further lowering of the substrate concentration was limited by low reproducibility of initial velocity measurements. RESULTS Pyruvate as Variable Substrate It has been well established that, according to Cleland s nomenclature [ 121, the lactate-dehydrogenase-catalyzed reaction follows an ordered bi-bi mechanism [13,14] with NADH the first and pyruvate the second substrate for binding to the enzyme. For such a mechanism, the steady-state initial rate can be expressed as Pyruvate (mm) I/NADH (mm- ) Fig. 1. Double-reciprocal plot and derived plots with pyruvate us the variable substrate. (A) Plots of reciprocals of initial reaction rates (a) versus reciprocal concentrations of pyruvate. The concentration of NADH was held constant at (0) 100 pm, (0) 25 pm, (H) 12.5 pm, (0) 9.1 pm, (A) 7.1 pm. (--) Lines from experimental data; (----) extrapolations of the linear portions of the experimental data at low concentrations of pyruvate. ti is expressed as NAD produced in the reaction mixture after addition of enzyme. (B) The plot of A versus pyruvate concentrations. A is obtained by subtracting the experimentally determined data of l/v from that obtained by linear extrapolation in (A). NADH concentrations were (0) 7.1 pm, (W) 9.1 pm. (C) The plot of A versu~ reciprocal values of NADH concentrations. A is the same as described in (B). [Pyruvate] = 1 mm indicates that the Michaelis-Menten constant and the dissociation constant of the enzyme. NADH complex are equivalent. Therefore, Eqn (1) can be converted to : where [A] and [B] are concentrations and K, and KI, are Michaelis-Menten constants of NADH and pyruvate, respectively. Ki, is the dissociation constant of the enzyme. NADH complex. The initial and maxima1 reaction rates are VI and V, respectively. The double-reciprocal plot using pyruvate as the variable substrate in the presence of five concentrations of NADH is shown in Fig. 1 A. The linearity of the plots at low concentrations of pyruvate suggests that the kinetic property of lactate dehydrogenase follows Eqn (1). The convergence of the lines on the abscissa calculated from the negative reciprocal of the intercept at the abscissa, was 71 pm. At higher concentrations of pyruvate, however, the Lineweaver-Burk plot was not linear. If one assumes that pyruvate forms an inactive binary complex with the enzyme, Eqn (2) can be converted to [I + ~ v K, P I +- ( 2)++ + = (1 + E)] (3)

3 C.-S. Wang 571-3o c B -ee?r TI /Pyruvate (rnm-') Pyruvate (mm) Fig. 2. Double-reciprocal plot and derived plots with NADH as the variuhle substrate. (A) The plot of reciprocals of initial reaction rates (v) versus reciprocal concentrations of NADH. The concentration of pyruvate was held constant at (A) 1000 pm, (0) 250 pm, (0) 125 pm. (m) 71.4 pm, (0) 50 pm, (A) 33.3 pm; u is expressed as NAD' produced in the reaction mixture after addition of enzyme. (B) The plot of Ki (the negative reciprocals of the intercepts at the abscissa); obtained from (A) versus pyruvate concentrations. (C) The plot of the ordinate intercepts from (A) versus the reciprocals of the pyruvate concentrations. (D) The plot of the slopes from (A) versus pyruvate concentrations +) 2 [ I +- Kb + 3 (1 + (1 + q ( 4 ) V P I [A1 where Klb is the dissociation constant of the inactive binary complex of the enzyme and pyruvate. The initial reaction rate, 212, is a complex function of the NADH and pyruvate concentrations. Since it is difficult to evaluate the inhibitory effect of pyruvate from the nonlinear curve of the Lineweaver-Burk plot (Fig. 1 A and Eqn 3), I have introduced a parameter defined as the difference between l/v~ and l/ul. The new parameter A, obtained by subtracting Eqn (2) from Eqn (3), is a linear function of both pyruvate concentrations and the reciprocals of NADH concentrations as shown by 1 1. (5) By inspecting the area of inhibition in Fig. 1 A the numerical values for A can be obtained by measuring the differences between the experimentally determined reciprocals of the initial rates and those obtained from the extrapolated linear plot. When A was expressed as a function of the pyruvate concentrations or reciprocals of the NADH concentrations (Fig. 1 B, C), both plots yielded linear relationships within experimental error. These results clearly indicate that the inhibitory effect of pyruvate is compatible with the competitive mechanism of substrate inhibition. NADH as Variable Substrate A double-reciprocal plot of the initial velocity data with NADH as the variable substrate in the presence of six concentrations of pyruvate gave lines with no common point of convergence (Fig.2A). The negative reciprocals of the intercepts at the abscissa of Fig.2A were defined as the apparent Michaelis- Menten constants of NADH, K,'. By inspection of Eqn (4), a linear relationship between K,' and pyruvate concentration can be predicted as shown in Eqn (6) : \ J Fig.2B shows that the data are compatible with Eqn (6). From the intercept and the slope determined from Fig. 2B, the Michaelis-Menten constant, K,, and the dissociation constant of the enzyme. pyruvate binary complex, Klb, were found to be 7.1 pm and 101 pm, respectively. A plot of the intercept of the ordinate of Fig.2A against the reciprocals of the pyruvate concentrations yielded a straight line (Fig. 2 C). The Michaelis-Menten constant of pyruvate can be calculated from the reciprocals of the intercept at the abscissa. The value was found to be

4 512 Competitive Substrate Inhibition formational change. As shown in Fig.lB, such a linear plot was obtained for lactate dehydrogenase with concentrations up to 1 mm of pyruvate. The plot of the parameter A versus the reciprocals of the concentrations of the cofactor can also be used to distinguish between the competitive and uncompetitive mechanisms of substrate inhibition. For the competitive Eqn (7) and uncompetitive Eqn (8) substrate inhibitions, in the case of the ordered bi-bi reaction, the parameter A can be expressed as a function of the substrate concentrations as shown below : IINAD' (rnm-') Fig. 3. Plot ofreciproca1.p of initial reaction rates (v) versus reciprocal Concentrations qf NAD +. The concentration of NADH was held constant at (0) 0, (m) 6.1 pm. [Lactate] = 0.5 mm similar to that obtained from Fig. 1 A. The slopes of the linear sections of the curves of Fig.2A when plotted against the pyruvate concentrations yielded a hyperbolic curve (Fig. 2 D) in accordance with that indicated by Eqn (4). Determination of Ki, The double-reciprocal plot of the data for the reverse reaction of lactate dehydrogenase using NAD + as the variable substrate in the presence of NADH is shown in Fig.3. The intersecting nature of the lines upon the ordinate axis indicates that NADH is a competitive product inhibitor against + NAD. Calculation of the dissociation constant of the enzyme. NADH binary complex, Ki,, was made from the change in slope resulting from the presence of NADH. This change yielded a Ki, value of 6.8 pm which is essentially the same as the previously determined K, of 7.1 pm. The conversion of Eqn (1) to Eqn (2) is, therefore, further supported by the similarity of the experimentally determined Michaelis-Menten constant and the dissociation constant of the enzyme. NADH complex. DISCUSSION The present kinetic study shows that the experimental estimation of parameter A is a very useful new method for distinguishing between the various mechanisms of substrate inhibition. If the parameter A is a linear function of the inhibitory substrate concentration, the maximal reaction rate V remains constant and the inhibition is not caused by a con- Kib is the dissociation constant of the second substrate B and the corresponding enzyme form. The parameter A, as expressed in Eqn (7), is a linear function of 1/[A] for competitive substrate inhibition. On the other hand, the parameter A should remain constant with the variation of [A] as expressed in Eqn (8) for uncompetitive substrate inhibition. This fact can be utilized for differentiating between these two types of substrate inhibitions. Since Cleland [15] has previously indicated that the inhibitory effect of substrate caused by the abortive complex formation can be considered as uncompetitive [ 1.51, the observed linear relationship between the parameter A and the reciprocals of NADH concentrations (Fig. 1 C) rules out the abortive complex formation as the basis for the inhibitory effect of pyruvate. The competitive nature of this inhibition can be further supported by the observed kinetic patterns with NADH as the variable substrate. The two distinct features of the Lineweaver-Burk plot as shown in Fig.2A are in accordance with those indicated by Fromm [16]. First, the intercept at the ordinate decreased as pyruvate concentration increased despite the inhibitory effect of pyruvate. A replot of the ordinate intercepts versus the reciprocals of the concentrations of pyruvate yielded a straight line (Fig. 2C) in accordance with Eqn (4). The second feature of the kinetic pattern of Fig.2A is the hyperbolic relationship between the slopes obtained from the linear plots and pyruvate concentrations. It can be deduced from Eqn (4) that the slopes become minimal when the concentration of pyruvate is l'm or 85 pm. The curve obtained from replotting the slopes versus pyruvate concentrations (Fig. 2 D) appears to be consistent with that predicted in Eqn (4). It has been shown by product inhibition patterns [I71 and spectroscopic evidence [ 11 that the abortive complex can occur if NAD+ is also added initially to the assay mixture. However, under the present assay conditions for human erythrocyte lactate dehydrogenase, the

5 (2.3. Wang 573 observed competitive nature of substrate inhibition clearly ruled out the abortive complex formation as a basis for the inhibitory effect of pyruvate. Examination of the parameter A as a function of substrate concentrations indicates that it cannot be utilized to differentiate competitive from non-competitive substrate inhibition. However, the Lineweaver- Burk plot (Fig. 2A) indicates that, despite the inhibitory effect of pyruvate, an increase of pyruvate concentration always results in an increase in apparent V. Therefore, the effect of pyruvate is not due to noncompetitive substrate inhibition. If a contaminating inhibitor of the tautomeric isomer enol-pyruvate was present in the pyruvate preparation, its inhibitory effect should not be a function of NADH concentration, which is different from the results shown in Fig.1C. Since the equilibrium constant for the formation of the tautomer enolpyruvate is low [18] and the pyruvate inhibitory effect is NADH-dependent, the presence of a contaminating inhibitor in the pyruvate preparation is an unlikely cause for substrate inhibition of lactate dehydrogenase. Similarly, if NAD+ or an NAD+-like inhibitor were present in the NADH preparation, the inhibition of lactate dehydrogenase should increase with an increased concentration of NADH. However, data in Fig. 1 C show that the inhibition of lactate dehydrogenase actually decreased with an increase in the concentration of NADH. Therefore, the presence of contaminant inhibitors in the NADH preparation as a basis for the inhibitory effect of pyruvate was also excluded. Although a competitive interaction between two substrate molecules has been proposed in theory as a possible mechanism of substrate inhibition [8,16], to my knowledge such an inhibitory mechanism has not been shown in any known enzymic system. The results of this kinetic analysis for lactate dehydrogenase are compatible with the proposed mechanism of competitive substrate inhibition. The data indicate that the inhibitory effect of pyruvate on lactate dehydrogenase is highly dependent on NADH concentration. When pyruvate is present in high concentrations, it apparently binds the free enzyme first and prevents the binding of NADH at a proper orientation for the catalytic reaction to occur. Alternatively, the initial binding of pyruvate may block the active site and prevent the cofactor from entering to its binding site. Although the estimation of the parameter A is straight-forward, as indicated in Fig. 1, some comments on its measurements deserve to be mentioned. At high concentrations of substrate A, an experimentally reliable linear Lineweaver-Burk plot can be obtained which facilitates the measurement of A. However, competitive substrate inhibition is observed only at the higher concentrations of substrate B. At low concentration of substrate A, the linear portion of the Lineweaver-Burk plot is, in some cases, difficult to obtain and the measurement of A becomes difficult. Therefore, a choice of optimal concentrations of A and B is necessary for proper measurement of the parameter A. The derivation of the kinetic constants K, and Klb was based on the special case where K, = Ki,; in this case the apparent Michaelis-Menten constant K,' is a linear function of [B] as shown in Eqn (6). However, when K, # Ki,, K; becomes a non-linear function of [B] [Eqn (9)] : To determine K, and KIb in this case, Eqn (9) is rearranged so that [B] becomes related linearly to the function Kd [l + (Kb/[B])]/[l + (KiaKb/Ka[B])] [Eqn (1011: The terms Kia/Ka and Kb can be obtained from Fig. 1 A and Fig. 2 C. The inhibitory effect of pyruvate on lactate dehydrogenase has not been observed under physiological conditions [ Therefore, substrate inhibition of lactate dehydrogenase has been suggested to be a phenomenon which only occurs in vitro. However, previous studies might have neglected the importance NADH plays in the expression of the pyruvate inhibitory effect. The results of this study indicate that inhibition of lactate dehydrogenase by pyruvate is a reciprocal function of NADH concentration, where pyruvate exerts an inhibitory effect on the enzyme only when the [NADH]/[pyruvate] ratio is low. The apparent physiological role of such a phenomenon could be to preserve a level of NADH concentration necessary for other vital enzymic reactions of living cells despite the presence of a high concentration of pyruvate. It would be interesting to establish whether such a substrate inhibition mechanism is a general mechanism for pyridine-nucleotide-dependent dehydrogenases to regulate the cofactor concentration in living cells. I would like to thank Dr P. Alaupovic for his interest and support ofthis work. I am also grateful for the valuable criticism and suggestions from Drs E. Niday, M. Griffin and R. Delaney. I thank Mr R. Hatcher for his skilful technical assistance and Mr R. Burns and Mrs M. Farmer for their help in the preparation of the manuscript. This study was supported by the resources of the Oklahoma Medical Research Foundation.

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