Inhibition of Human Erythrocyte Lactate Dehydrogenase by High Concentrations of Pyruvate. Evidence for the Competitive Substrate Inhibition

Abstract

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 + and 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

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

“…The absence of linearity for SAV UM , SAV DEX , and SAV FIC (Fig. 1F) indicates that competitive binding of substrate to the active-site is not the cause of inhibition at high substrate concentrations (Wang, 1997). The markedly undulating curves, particularly for SAV UM (Fig.…”

“…The kinetics were further analyzed by the D method of Niday et al (1980) and Wang (1997). Lineweaver-Burk plots (Fig.…”

“…The data were fitted directly to Michaelis-Menten plots (Kato et al, 1972) and ranked according to various kinetic models using the Enzyme Kinetics Module 1.1 linked to Sigma Plot 8.02. The data were also plotted using the D method of Wang to further define the type of substrate inhibition (Wang, 1997).…”

Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement

“…In order to increase the activity of 6-PGDH, and hence lower the simulated in i o steady-state concentration of 6- [87] showed that the allosteric and co-operative properties of PK are strongly influenced by pH and this suggested that the transition from R to T involves a deprotonation ; examination of the values that these workers obtained for L at different pH values shows that they are most closely matched if it is assumed that n l 1 (to the nearest integer) ; the pH-dependence of the inhibition of erythrocyte PK by iodoacetamide involves a group that has a pK a of 6.8 [88] ; it was assumed that this was the group that was involved in the R-to-T transition. [89] adjusted to 37 mC using the van 't Hoff equation ; d K eq l Lac NAD/(NADH Pyr) ; e k cat,f was calculated from the specific activity of the partially purified human erythrocyte enzyme (400 units/mg at pH 7.4, 37 mC ; [90]) ; the M r of the tetramer was taken to be 144 000 (see [90]) ; the specific activity of the forward reaction was found to be 4.7 times that of the reverse reaction [91] ; f the concentration of LDH in human erythrocytes, e o , was estimated from the measured human erythrocyte activity (66000 units/litre of erythrocytes, pH 7.6, 37 mC ; [62]) and the value of k cat,f . PG, the K m values of NADP + and 6-PG were decreased by a factor of 2, while the K m values for Ru5P and NADPH were increased by a factor of 2.…”

Impact of High Pyruvate Concentration on Kinetics of Rabbit Muscle Lactate Dehydrogenase

“…The ternary formation has become better known as an E-NAD + -Pyr adduct complex [2][3][4][5]. Additional work promoted the idea of simple enzyme-pyruvate binary complexes and other inhibiting ternary complexes involving only cofactors [6,7]. Recently, the substrate binding has been examined with extremely small timescales to better understand the enzyme's intricate mechanics [8].…”