Heart, lactate dehydrogenase

The ability of unsaturated ketonucleosides to react with protein sulf-hydryl groups was demonstrated by measuring their inhibitory action towards beef-heart lactate dehydrogenase.75 These results led to the conclusion that the primary targets for unsaturated ketonucleosides are glutathione and reactive thiol groups of proteins. 

Figure 9.8. The spectral density Cs(co) for the reaction coordinate in the wild type human heart lactate dehydrogenase isoform. The solid line represents the configuration where lactate and NAD+ are bound, the dotted line is when pyruvate and NADH are bound, the dashed line is the minimal coupling (MC) simulation with lactate and NAD+ bound and the...

The catalysis of the oxidation of aldehydes to carboxylates by alcohol dehydrogenases raises questions regarding the function of the active site thiols found in most aldehyde dehydrogenases. Clearly a free thiol is not mechanistically essential for aldehyde oxidation. For example, pig heart lactate dehydrogenase catalyzes the facile oxidation of glyoxalate to oxalate (71), glucose-6-... 

The initial rate equation is again of the form of Eq. (1) with the kinetic coefficients as in Table I, which shows that the mechanism differs from the simple ordered mechanism in three important respects. First, the isomerization steps are potentially rate-limiting evidence for such a rate-limiting step not attributable to product dissociation or the hydride-transfer step (fc) has been put forward for pig heart lactate dehydrogenase 25). Second, Eqs. (5) and (6) no longer apply in each case the function of kinetic coefficients will be smaller than the individual velocity constant (Table I). Third, because < ab/ a< b is smaller than it may also be smaller than the maximum specific rate of the reverse reaction that is, one of the maximum rate relations in Eq. (7) need not hold 26). This mechanism was in fact first suggested to account for anomalous maximum rate relations obtained with dehydrogenases for which there was other evidence for an ordered mechanism 27-29). 

In this mechanism, the K values ( a/ o, b/ o) are dissociation constants for the ternary complex. Equation (8) holds, and in addition 4>AB/A = Keb and pq/0p = Keq- The mechanism was early rejected for beef heart lactate dehydrogenase on the grounds that enzyme-lactate and enzyme-pyruvate compounds having these dissociation constants... 

The large values of a b/ ab o for yeast alcohol dehydrogenase with butanol as substrate, for bovine heart lactate dehydrogenase at pH 6.0,... 

The interpretation and value of reversible inhibition kinetics have been set forth clearly by Cleland (8,18). The uses of product inhibition studies, particularly for distinguishing random and ordered mechanisms, was suggested by Alberty (7) and first applied and developed by Fromm (58,83). Schwert and his colleagues first gave a detailed theoretical and experimental analysis of inhibition by substrate analogs in their important work on oxamate and oxalate inhibition of bovine heart lactate dehydrogenase (84) ... 

The constancy of Ae.nad/Ae.nadh at pH 6.0-8.5 for other dehydrogenases in Table IV, notably the heart lactate dehydrogenases, means that a proton is released stoichiometrically in the reduction of E NAD to E NADH by substrate, just as in the overall reaction [Eq. (11)]. A histidine residue as proton source and sink was first suggested by Schwert et al. 56,67) and can now be identified with the single essential histidine per subunit 168) in the pig heart enzyme and histidine-195 in the dogfish enzyme. The constancy of Ae nad/Ae.nadh shows that the... 

Fortunately, as the possible mechanisms proliferate, the analytical power of enzyme kinetics increases, since the kinetic patterns lead to diagnostic tests for various mechanisms. The task of unravelling 2- and 3-substrate mechanisms began in earnest in the 1950 s with the theoretical framework laid by Alberty [45,46], Dalziel [47,48] and Cleland [49-51]. The detailed experimental investigations of horse liver alcohol dehydrogenase by Theorell, Dalziel and their collaborators [42,52] and of beef heart lactate dehydrogenase by Schwert s group [53] were important milestones. 

Grau UM, Trommer WE, Rossmann MG (1981) Structure of the active ternary complex of pig heart lactate dehydrogenase with S-lac-NAD... 

Recently a Cl NMR line width study of another metal-free enzyme, pig heart lactate dehydrogenase, has been presented by Ward [48Z]. Two types of Cl binding sites were inferred, one of which is... 

A very simple derivative of nicotinamide, 3-bromoacetylpyridine, preferentially alkylates both an essential cysteinyl and an essential histidyl residue in pig heart lactate dehydrogenase. The reagent is selective for the histidyl residue if the enzyme is pretreated with mercuric ions to protect the sulfhydryl group. Subsequent to these investigations the three-dimensional structures of dogfish lactate dehydrogenase and... 

Although, favorable factors are present, the system prefers to remain aromatic. Hence, the formation of NADH in the enzymatic system could be driven by conformational changes that shift the equilibrium toward the nonaromatic species. However, in 1978, a German group (276) observed an intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase using a coenzyme-substrate covalent analogue composed of lactate and NAD+. 

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... 

Figure 5.4 The reaction of pig heart lactate dehydrogenase with lactate and NAD. The four panels show (a) two phases of NADH formation (enzyme bound and free), (/>) NADH fluorescence, which is predominated by the enzyme-NADH complex after pyruvate dissociation, (c) protein fluorescence quenching monitoring concentration of all enzyme-NADH complexes and (d) phenol red absorbence monitoring two phases of proton liberation. (For detail see text and Whitaker ef n/., 1974.)...

Fig. 5.22. Influence of pH and temperature on the reactivation of pig heart lactate dehydrogenase (from Jaenicke, 1974).

Fig. 5.24. Effect of protein concentration on the reactivation and reassociation of pig heart lactate dehydrogenase (LDH-H4) (from Jaenicke, 1974). Enzyme was deactivated at pH 2.3 and reactivated in 0.2 M phosphate buffer pH 7.95 + 1 mM DTT +10 mM EDTA at 20°C for 3 hr (A) 20-hr ( ) yields of the tetrameric species obtained from centrifugation velocity (O) (o), reactivation after denaturation in 6 M GuHCl (courtesy of Jaenicke).

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