Kinetic studies of rabbit muscle lactate dehydrogenase.

A number of reports have appeared in recent years on the mechanism, i.e. the mode of enzyme and substrate interaction, of anaerobic dehydrogenases. For example, the mechanisms of heart muscle lactate dehydrogenase (1)) yeast alcohol dehydrogenase (2), and ribitol dehydrogenase (3) appear to involve a compulsory sequence of enzyme and substrate interaction leading to one or more kinetically important ternary complexes. In 1951 Theorell and Chance (4) formulated a reaction sequence for liver alcohol dehydrogenase which involves only binary complexes; however, more recent studies indicate that whereas ternary complexes may be formed in the liver alcohol dehydrogenase reaction, they are short-lived relative to the binary complexes involving enzyme and coenzyme (5, 6). Diand triphosphopyridine nucleotide-linked dehydrogenase enzymes lend themselves conveniently to kinetic studies, as the concentration of reduced coenzyme can be measured simply and precisely. In most investigations, however, a number of mechanisms were found to be compatible with dehydrogenase kinetic data (7). In such instances it was necessary to correlate the dissociation constants determined kinetically with such constants evaluated by other means (8, 9j. These latter analyses require a homogeneous preparation of enzyme of known molecular weight. In studying reversible reactions kinetically, it has proven advantageous to employ the Haldane relationship as suggested by Alberty (10). This method was recently utilized to exclude the “Theorell-Chance” mechanism (4) in the case of ribitol dehydrogenase (11). It was observed that there was a 50% discrepancy between the apparent equilibrium constant and this constant determined kinetically, and for this reason, the binary complex mechanism was omitted from consideration for ribitol dehydrogenase. Subsequent experiments, in which a different approach was used, appeared to establish the correctness of this view (3). On the other hand, Theorell, Nygaard, and Bonnischen (5) reported good agreement between the Haldane equation for the binary complex mechanism and poor correlation between this equation and the ternary complex pathway of enzyme and substrate interaction. The usefulness of studying reaction mechanisms in the absence and presence of product has very recently been suggested (3, 12). The steady state equations put forth to account for product inhibition include the possible formation of enzymereduced coenzyme-reduced product and enzyme-oxidized coen-