Lactate dehydrogenase dissociation constants

Colorimetric and fluorimetric NH3 sensors contain mixtures of pH indicators having suitable dissociation constants at the tip of the fiber bundle. The measuring solution is separated from this indicator layer by an NH3 gas-permeable membrane covered by an immobilized de-aminating enzyme, e.g. urease (Wolfbeis, 1987 Arnold, 1987). The fluorimetric indication of NADH has been used in optical biosensors for lactate, pyruvate, and ethanol, where the respective dehydrogenase is immobilized at the tip of an optical NADH sensor (Arnold, 1987 Wangsa and Arnold, 1988). 

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 results of stopped-flow studies of the lactate dehydrogenase reaction have proved to be more difficult to interpret than those of alcohol dehydrogenase. The dissociation velocity constants for the binary NADH compounds of the pig heart and skeletal muscle lactate dehydrogenases are much larger than that for liver alcohol dehydrogenase, and also larger than the maximum specific rates of lactate oxidation at pH 6.0-7.0 (Table VII). Some earlier step must therefore be rate-limiting. 

It is important to point out that the values of the rate constant for the dissociation of a ligand from its specific binding site, obtained by the two above methods (displacement and transfer), are not necessarily the same. Wu et a/. (1991) noted that the rate constants for NADH dissociation from its complexes with lactate dehydrogenase and glycerol-3-phosphate dehydrogenase are more than 50% higher when measured by ligand displacement... 

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