Determination of Dissociation Constants of Coenzymes and Abortive Ternary Complexes with Rabbit Muscle Lactate Dehydrogenase from Fluorescence Measurements.

Studies on the mechanism of substrate interaction with rabbit muscle lactate dehydrogenase have recently been reported (1). These investigations, which were primarily kinetic in nature, indicated that nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide must add to the enzyme before the binding of substrates pyruvate and L-lactate. It was also noted that ternary complexes of lactate dehydrogenase, coenzymes, and substrates are short-lived relative to dissociation of the binary complexes. Many of the definitive conclusions alluded to in the earlier study (1) were made on the basis of kinetic experiments carried out with and without product (2,3). Equations derived to evaluate such experiments assumed the formation of abortive ternary complexes, enzyme-NAD-pyruvate and enzyme-NADH-n-lactate (1). Ternary complexes of these types have been assumed for other enzyme systems from kinetic (1, 2,4, 5), spectrophotometric (6), and spectrofluorometric studies (7-9). The possible formation of abortive ternary complexes in kinetic studies of product inhibition and their effect has recently been suggested (1,2,4) It is obviously desirable to correlate various parameters obtained from kinetic experiments with those determined by other means. In the latter case, a homogeneous enzyme preparation of known molecular weight is a prerequisite for such determinations. Hayes and Velick (10) were able to measure certain dissociation constants for glyceraldehyde a-phosphate dehydrogenase by employing the technique of high speed centrifugation. Takenaka and Schwert (11) utilized a similar procedure in the case of heart muscle lactate dehydrogenase. More recently, investigators have taken advantage of alterations in the fluorescence spectra of enzymes and substrates to evaluate dissociation constants of binary and abortive ternary complexes (7-9, 12, 13). The purpose of the present report is to present data obtained from fluorescence experiments of rabbit muscle lactate dehydrogenase. The results lend support to the mechanism of substrate and enzyme interaction proposed earlier (1).