Kinetic studies on human lactate dehydrogenase isoenzyme-catalyzed lactate-to-pyruvate reaction

In order to evaluate the functional differences that may exist in human lactate dehydrogenase (LDH) isoenzymes widely used for clinical examination the kinetic and thermodynamic properties of the lactate to pyruvate reaction that they catalize were examined. Small but significant differences in the kinetic properties of the three isoenzymes were observed. The difference in the rate constants might affect the activity measurement of the individual isoenzyme as the initial velocity for the L-P reaction catalyzed will not be the same for an equal amount of enzyme. Equilibrium constants for the overall reaction in the presence and absence of pyruvate have been determined. On the basis of transition-state theory, the standard enthalpy and free-energy changes for formation of ternary activated complex were positive, while the standard entropy change was negative. Although the standard free-energy change was the same for activation by the three isoenzymes, the enthalpy and entropy changes for the LDH-3-catalyzed reaction were different from the respective values for others. A large positive value for the free-energy change and a negative value for the entropy change indicated unfavorable production of the activated complex (K _infeq. ^sup╪ =1.89×10^-16). The enzyme appears to stabilize and retain the activated complex until it dissociates into the products.     

The initial reaction velocities of lactate dehydrogenase in various cell types

Summary The initial reaction velocities (v _v ) of lactate dehydrogenase in hepatocytes, cardiac muscle fibres, skeletal (gastrocnemius) muscle fibres, gastric parietal cells, ductal epithelial and acinar cells of the parotid gland, and oocytes were determined, by computer-assisted image analysis, in unfixed sections of these tissues incubated at 37°C on substrate-containing agarose gel films. They were found to fit the equations v _i = a₁A (equation 1) and v _i – v = a₂A (equation 2) reported previously for mouse hepatocytes (Nakae & Stoward, 1993a, b), where v and A are, respectively, the gradients (or steady-state velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) of the finalreaction product on incubation times between 1 and 3 min, and a ₁ and a ₂ are constants. Both equations 1 and 2 fitted the observed v _i closely for mouse (a ₁ = 2.7, a ₂ = 2.2) and human (a ₁ = 3.0, a ₂ = 1.9) hepatocytes. However, equation 2 fitted the observed v _i better than equation 1 for mouse cardiac muscle fibres (a ₂ = 1.5), skeletal muscle fibres (a ₂ = 1.2), gastric parietal cells (a ₂ = 1.7), acinar (a ₂ = 1.4) and striated ductal (a ₂ = 2.2) epithelial cells of the parotid gland, and oocytes (a ₂ = 1.6). The values of v _i calculated from the two equations agreed with the observed v _i to within about 11%. They ranged from 105 mole hydrogen equivalents/cm³ cell/min units in hepatocytes to 24 units in parotid acinar cells, but for other cell types they were between 46 and 61 units. These are all considerably higher than values reported previously.     

High concentrations of guanidine chloride activate lactate dehydrogenase in low water media

The effect of guanidine chloride on the activity of bovine heart lactate dehydrogenase transferred to a system that was made with toluene, phospholipids, Triton X-100 and 3.8% water (v/v) was studied. The activity of the enzyme in the latter system was about 30 times lower than in standard water mixtures. In the low water system, 1.5 and 2.0 M guanidine chloride increased the activity by approximately 20 times. These concentrations of guanidine chloride caused complete inactivation of the enzyme in conventional water systems. The activating effect of the denaturant was independent of enzyme concentration. It is suggested that the increase in activity produced by guanidine chloride was due to a facilitation of the protein-solvent interactions that operate in a catalytic cycle.     

Dynamic aspect of kinetics of reaction catalyzed by lactate dehydrogenase

The influence of solvent viscosity on the kinetic parameters of the pyruvate reduction reaction catalyzed by lactate dehydrogenase has been investigated. The viscosity was adjusted by sucrose and glycerol solutions at concentrations from 0 to 44% and from 0 to 63%, respectively. The reaction rate decreased abruptly with an increase in viscosity. The study of different reaction stages (enzyme-substrate complex formation, catalysis, inhibitory complex decomposition, competitive inhibition by chlorine ions) revealed that the catalysis (and the related conformational changes) is the only stage (of the above mentioned) that depends markedly on the solvent viscosity. The reaction is insensitive to the changes in the dielectric properties of the solution induced by the addition of alcohols and dioxane. The observed power dependence of the rate constant on viscosity is explained in terms of Kramer's theory which considers the proton transition through the activation barrier to be a diffusion in the field of random forces. The influence of solvent viscosity on enzymic kinetics indicates a direct relation between solvent dynamics and relevant protein conformational movements.     

Kinetics of lactate dehydrogenase and other enzyme studies in human serum in leukemia.

The activity of some of the clinically important enzymes was investigated in leukemic sera at 37 degrees, using the Beckman Enzyme Activity Analyzer were found to be slightly elevated in some untreated cases of leukemia (1.), while ALP was found to be frequently elevated. Untreated patients with l. had normal or below normal SCPK activity. The most characteristic and significant rise in activity, was found to be associated with SLDH and SHBDH in most cases of acute l. (86%) and in CML, while any elevation observed in CLL, was very slight. The general kinetic parameters of SLDH and SHBDH, were investigated at 37 degrees in acute leukemic patients. These included optimum substrate concentrations (NADH, pyruvate, and 2-oxobutyrate), the rate of pyruvate and 2-oxobutyrate reduction, substrate-velocity relationship, Km (pyruvate), Km (NADH), Km (2-oxobutyrate) as well as the effect of temperature and pH on the kinetics of the reaction. These kinetic characteristics were found to be differently affected by the leukemic process.     

Glutamate dehydrogenase of lupin nodules: Kinetics of the deamination reaction

The reaction of glutamate dehydrogenase (l-glutamate: NAD⁺ oxidoreductase (deaminating) EC 1.4.1.2) from lupin nodules has been investigated in the direction of deamination by means of steady state velocity studies in the absence of products and inhibition studies with products and substrate analogs. The results are qualitatively and quantitatively consistent with a fully ordered reaction mechanism in which NAD⁺ binds to the enzyme first followed by l-glutamate. The order of product release is proposed to be NH₄⁺ followed by 2-oxoglutarate and then NADH. In addition, product inhibition data indicate the formation of an enzyme-NAD-oxoglutarate dead-end complex.     

Autophosphorylation of lactate dehydrogenase

Incubation of rabbit muscle lactate dehydrogenase in the presence of Mg[alpha-32p]ATP results in the incorporation of the label into the protein. The autophosphorylation reaction is strongly pH-dependent. The maximal phosphorylation is observed at pH 6.8 with 3-4 moles of phosphate bound per mole of tetrameric enzyme. The enzyme-phosphate complex is readily hydrolyzed by hydroxylamine.     

Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution.