Fast‐Kinetic Studies of the Oxidative Deamination of Glutamate Catalyzed by Glutamate Dehydrogenase

Open Access
Publisher Website
Google Scholar
Abstract
The release of protons, one of the products of the reaction, in the oxidative deamination of glutamate by glutamate dehydrogenase, has been followed in a stopped‐flow spectrophotometer by measuring the absorbance change of an indicator dye. The results obtained at pH 7.8 and 15 °C, in 0.05 M Na2SO4, with NADP+ as coenzyme, indicate that the liberation of the protons occurs after the electron transfer but before the release of NADPH, i.e. before the steady state; the results obtained in the same conditions, but in presence of the inhibitor GTP, indicate moreover that it occurs also before the liberation of 2‐oxoglutarate.The oxidative deamination of glutamate has in addition been studied in the presence of equi‐molar amounts of the reduced coenzyme NADPH. The elimination of the presteady‐state step when all the enzyme is in the form of the enzyme · NADPH · glutamate complex supports the hypothesis that the rate‐limiting step in the overall mechanism is the dissociation of NADPH and the regeneration of the enzyme from this ternary complex.