The putative isomerization of the lactate dehydrogenase — NADH complex

Abstract

Transient kinetic studies of the mechanism of lactate dehydrogenase were helped considerably by the variety of optical signals available. This permitted the direct observation of the formation and interconversion of intermediates [l] . The formation of the binary complex, E. NADH, from free enzyme and reduced nucleotide can be observed both through the enhancement of nucleotide fluorescence and the quenching of protein fluorescence. Quantitative analysis of equilibrium [2] and kinetic [3] experiments indicated no distinction between binding of NADH to pig heart lactate dehydrogenase as monitored by the two signals, provided the correction for the nonlinearity of protein fluorescence quenching [2] is applied. Everse, Berger and Kaplan reported [4-61 that the reaction of reduced acetylpyridine adenine nucleotide with chicken-heart lactate dehydrogenase proceeds in two steps distinguishable through a second-order protein fluorescence signal and a first-order nucleotide fluorescence signal. The process monitored by nucleotide fluorescence was reported to have a rate constant of about 95 s-l and this was interpreted in terms of a first-order isomerisation, following a second-order binding process. We therefore re-examined the kinetics of the reaction of the pig heart enzyme with NADH to see whether we could detect a rearrangement with this species.