Lactate dehydrogenase equilibrium constants

The now classical example is lactate dehydrogenase. Sil-verstein and Boyer were the first to determine the rates of exchange between cognate pairs of reactants ie., lactate and pyruvate as well as NAD and NADH). Convenient [NADH]/[NAD ] and [pyruvate]/[lactate] ratios were chosen such that when combined they satisfied the apparent equilibrium constant for the LDH reaction. These investigators first established that each exchange rate was directly proportional to the duration of exchange and likewise directly proportional to enzyme concentration. As an additional control, they also demonstrated the equality of the pyruvate lactate exchange... 

The ratio [NAD+]/[NADH] appears to be maintained at a relatively constant value and in equilibrium with a series of different reduced and oxidized substrate pairs. Thus, it was observed that in the cytoplasm of rat liver cells, the dehydrogenations catalyzed by lactate dehydrogenase, sn-glycerol 3-phosphate dehydrogenase, and malate dehydrogenase are all at equilibrium with the same ratio of [NAD+]/[NADH].166 In one experiment rat livers were removed and frozen in less than 8 s by "freeze-clamping" (Section L,2) and the concentrations of different components of the cytoplasm determined167 the ratio [NAD+] / [NADH] was found to be 634, while the ratio of [lactate]/[pyruvate] was 14.2. From these values an... 

L-Lactate dehydrogenase (l-LDH, EC 1.1.1.27) catalyzes the reduction of pyruvate to (S)-lactate with a simultaneous oxidation of NADH. l-LDH is found in all higher organisms. There are two kinds of l-LDHs enzymes from one group are activated by fructose 1,6-diphosphate while the other group stays independent [71]. l-LDH is highly selective for pyruvate, short-chain 2-keto acids and phenylpyruvic acid [80]. All bacterial NAD+-dependent LDHs form lactate from pyruvate in vivo, and there is no evidence at all that they catalyze the other direction as well. The equilibrium constant lies far on the direction of lactate formation, and thus the reaction catalyzed by bacterial LDHs can be considered almost irreversible. LDHs from some lacto-bacilli like Lactobacillus fermentum or L. cellobiosus show no or just poor reaction with lactate [71], whereas mammalian LDHs can be considered as reversible [71]. Well characterized l-LDHs are summarized in Table 2. 

Enzyme-catalyzed reactions do not always proceed to completion because of an unfavorable equilibrium constant or insufficient reaction time. Nevertheless, a compound can be determined by a total change method. The reaction may be pulled to completion by trapping the products or by coupling one of the products to a second enzyme reaction. In determining lactate with lactate dehydrogenase the equilibrium lies far to the left in the direction of lactate at pH 9.5 (K = 2.9 X 10"at 25 C). 

Under certain conditions, the ratio of lactate to pyruvate is an indicator of redox status. By rearranging the equation for the equilibrium constant for the reaction catalyzed by lactate dehydrogenase (EC 1.1.1.27), it can be seen that the ratio of lactate to pyruvate is proportional to the ratio of NADH to NADL... 

There have been extensive discussions in the literature regarding maximization of the catalytic efficiency of enzymes and the value of their internal equilibrium constants is the equilibrium constant between substrates and products of the enzyme when all are bound productively) (98-102). For example, the value of A int is near unity for both liver alcohol dehydrogenase (78) and lactate dehydrogenase (103) when measured with their natural substrates. The ability of these same enzymes to function with alternative substrates with widely differing external equilibrium constants raises important questions regarding the relationships of the internal thermodynamics of such reactions. 

In chapter S the phenomenon of on enzyme equilibria is discussed with examples. This refers to the fact that the equilibrium between enzyme-substrate and enzyme-product complexes is often near unity, even if the overall equilibrium constant for the interconversion of free substrate to free product is a large number. This does not contradict the statement that enzymes (or catalysts in general) do not affect equilibrium constants of reactions. It has to be remembered that this definition of catalysis only applies to the equilibrium between free substrates and products. An example, which illustrates this in terms of the Haldane relation, is heart lactate dehydrogenase. By the methods discussed in section 5.1 it was shown that the equilibrium constant for the two complexes... 

In several NAD linked dehydrogenases, ATPases and phosphokinases the equilibrium constant for step II, the interconversion of the enzyme-substrate to the enzyme-product complex is near unity (see Gutfreund Trentham, 1975 Edsall Gutfreund, 1983). The thermodynamic rationale for this phenomenon is also given in section 3.3 and it will be documented further when the reaction pathways of myosin ATPase and of lactate dehydrogenase are described below. 

The co-product pyruvate of the TA reaction is constantly removed by lactate dehydrc e-nase (LDH) or recycled back to alanine by alanine /tADH (AlaDH). This is done to push the equilibrium toward the amine. i-Alanine is added for the amination with (S)-oTAs and D-alanine is added for amination with (R)-coTAs. The cofactor NADH is regenerated by 11 U of C. boidinii formate dehydrogenase (CbFDH, Codexis). 

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