Macroscopic rate constants involved in the formation and interconversion of the two central enzyme--substrate complexes of the lactate dehydrogenase turnover

Abstract

The preceding paper (Südi, 1974) reports partial success in describing the conversion of E(NADH) plus pyruvate into E(NAD+) plus lactate in terms of a simple Haldane-type scheme which involves two intermediates (E(NADH) (Pyr) and E(NAD+) (Lac)), where E represents lactate dehydrogenase. This information is completed here by reporting kinetic results obtained by carrying out the same reaction in the opposite direction. The combined results of these two papers confirm the findings of Holbrook & Gutfreund (1973) that the observed spectral changes do take place at the level of resolution of this simple two-intermediate scheme. The following numerical values for the rate (and equilibrium) constants involved in their formation and decomposition are reported: [Formula: see text] It is shown that although the precision of estimation of some of these numerical values is subject to some experimental uncertainty, their derivation from direct experimental observations only involves the principle of microscopic reversibility. This paper describes stopped-flow kinetic observations made with E(NAD+) and lactate as the two reactants. It is shown that fluorescence and u.v.-absorption measurements yield the same experimental rate constant for the last reaction step in which E(NADH) is generated. On the other hand, the generation of E(NADH) (Pyr) can only be indirectly observed, as a less than stoicheiometric ;burst', and by u.v.-absorption measurements only. It is shown that the stoicheiometry of this partial ;burst reaction', and a pre-equilibrium factor in the directly observed rate of E(NADH)-production, yield equivalent information about the reversible oxidation-reduction step. It is further shown that the pre-equilibrium factor that is involved in the generation of E(NADH) can be determined because k(+4)=222s(-1) is already known (Südi, 1974). Since the fluorescence measurements yield much more precise estimations, and their interpretation is considered by the author to be free of ambiguity, the presented quantitative analysis is based on the fluorescence observations.