Kinetic studies of some mechanisms of lactate dehydrogenase with the tritium isotope effect

Abstract

The mechanism of action of rabbit‐muscle lactate dehydrogenase was studied earlier by Thomson with deuteriated NADH. Reexamination of the reverse reaction with tritium‐labelled [A‐4‐3H]NADH and study of the forward reaction with L‐[2‐3H] lactate require a new interpretation of the isotope effects.L‐[2‐3H]lactate yields an isotope effect of kH/k3H= 2.5 (25°) which decreases slightly with decreasing temperature. This is in agreement with the rate limiting influence of two steps: isomerisation of enzyme‐NADH complexes and NADH dissociation from this complex; both steps may cause secondary isotope effects. Their size may be compared to those in the rate limiting NADH dissociation of the liver alcohol dehydrogenase catalyzed reactions (1.3–1.8).The existence of an isotope effect in the reverse reaction (1.0–2.0 at 25°) excludes the ratelimiting enzyme‐coenzyme isomerisation or coenzyme dissociation found in the forward reaction. Despite the similarity in size between the deuterium and tritium NADH isotope effects, the latter effect is too small to support kinetically significant ternary complexes as proposed by Thomson. As in the forward reaction, the isotope effects are secondary effects, which can arise during NADH binding, enzyme‐NADH isomerisation, or eventually, lactate dissociation.The results are in agreement with an iso‐Theorell‐Chance mechanism as proposed by Zewe and Fromm and which indeed requires an influence of enzyme‐NADH isomerisation in the forward and reverse reaction.With increasing concentrations of pyruvate the isotope effect of the reverse reaction disappears, due to a now rate‐limiting dissociation of NAD from the inhibitory complex enzyme‐NAD‐pyruvate.