A kinetic study of the oxidative deamination of L‐glutamate by Peptostreptococcus asaccharolyticus glutamate dehydrogenase using a variety of coenzymes

Abstract

1. The NAD + -specific glutamate dehydrogenase from Peptostreptococcus asaccharolyticus follows MichaelisMenten kinetics in contrast to the enzyme from several other sources, and thus gives linear double-reciprocal plots of initial-rate data.2. The initial-rate parameters have been determined for this bacterial glutamate dehydrogenase in the direction of oxidative deamination. The use of alternative coenzymes leads to some conclusions about the order of substrate addition.3. An investigation of the pH dependence of this reaction reveals that the binding of oxidised coenzyme is independent of pH over the range 6 -9.4. The kinetic data are consistent with an ordered addition of coenzyme prior to glutamate, the reverse of the mechanism derived with ox glutamate dehydrogenase in the presence of ADP.Peptostreptococcus asaccharolyticus is an exceptionally rich source of glutamate dehydrogenase, which catalyses the following reaction : L-Glutamate + NAD' + HzO 2-oxoglutarate + NH:The enzyme is highly specific for L-glutamate [l], although the coenzyme specificity is somewhat broader. This bacterial dehydrogenase shares some features with the corresponding mammalian enzymes : it is hexameric, with a subunit molecular mass of 49000 Da. However, it is not regulated by the purine nucleotides ADP or GTP, and double-reciprocal plots for oxidised coenzyme follow Michaelis-Menten kinetics. A major complicating factor in steady-state kinetic studies of ox liver glutamate dehydrogenase has been the superimposed cooperativity with respect to both NAD' and NADP' [2]. This problem of non-linearity has also arisen in the study of several other glutamate dehydrogenases (e.g. [3 -51). The opportunity to study a glutamate dehydrogenase bi-ter mechanism with simplified behaviour is afforded by the enzyme from P. asaccharolyticus. Several alternative coenzymes are used here to elucidate the order of substrate binding in the two-substrate direction (oxidative deamination of glutamate). This approach has also been used recently to study the same reaction with the ox liver enzyme in the presence of ADP [6]. In that study the results were consistent with an ordered mechanism, in which glutamate is the leading substrate.As a second approach, the pH dependence of the oxidative deamination of glutamate was examined with NAD' as the coenzyme. The initial-rate parameters were determined for seven pH values, and certain ratios of these parameters shed Abbreviations. AcPdAD+, acetylpyridine-adenine dinucleotide;Enzyme. Glutamate dehydrogenase (EC 1.4.1.2).dnNAD', deamino-NAD' , some light on the mechanism of the reaction. This investigation we believe, represents the first example of a detailed pH study with glutamate dehydrogenase from any source and is analogous to the work of Winer and Schwert [7] on lactate dehydrogenase, and Dickenson and Dickinson [8,9] on yeast alcohol dehydrogenase. MATERIALS AND METHODSThe details of bacterial growth and enzyme purification have been described elsewhere [I, 101. The enzyme was stored as a suspension in 80...