The concentration dependence of the activity of beef liver glutamate dehydrogenase as measured by rapid mixing and ultracentrifugation techniques

Kempfle, M.; Mosebach, K.-O.; Winkler, H.

doi:10.1016/0014-5793(72)80065-6

Cited by 6 publications

(1 citation statement)

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“…Therefore, the association sites [48] must be different from the active sites. This is in agreement with the finding that the specific activity of this enzyme is independent of its state of association [39,49] although contradictory results have been published [50].…”

Section: Discussionsupporting

confidence: 92%

Studies of Glutamate Dehydrogenase

Krause

Bühner

Sund

1974

European Journal of Biochemistry

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The binding of NADH and NADPH to beef liver glutamate dehydrogenase has been investigated using fluorescence titration and the preparative as well as the analytical ultracentrifuge over a wide range of protein and coenzyme concentrations. The glutamate dehydrogenase oligomer (molecular weight 336 000, six polypeptide chains) binds independent of enzyme concentration a total of twelve molecules of each reduced coenzyme, NADH and NADPH. The first six binding sites, the "active sites", bind NADH and NADPH in the same manner, whereas the weaker binding to the other six sites, the "nonactive sites", is remarkably different for both coenzymes : probably due to the steric hindrance or electrostatic repulsion the affinity of the enzyme for NADPH is about 10 times lower than that for NADH. This result explains the different kinetic behaviour of the two coenzymes. From the results obtained at different protein concentrations it is concluded that the coenzyme binding sites and the association sites are located at different parts of the enzyme surface.A procedure is described for three-parameter fitting of titration curves using a programmable desk calculator with on-line x-y recorder.Glutamate dehydrogenase from beef liver catalyzes the reversible oxidation of glutamate to 2-0x0-glutarate with NAD as well as NADP as coenzyme. Some experimental data indicate that a t high coenzyme concentrations NADf acts as an activator and NADH as an inhibitor of the enzymatic reaction, whereas in the presence of NADP+ or NADPH in general a normal Lineweaver-Burk plot is observed [5]. This difference in kinetic behaviour and the rate of dissociation induced by the coenzyme in the presence of GTP were explained by assuming two distinct binding sites per polypeptide chain for NAD but only one such site for NADP [5,6]. However other kinetic studies [7,8] show no difference in principle between the two oxidized coenzymes. The oligomer is composed of six polypeptide chains with identical sequence [9] and from a chemical point of view it seems reasonable to assume an identical reaction mechanism for both coenzymes [lo], even if quantitative differences between corresponding kinetic parameters exist. This paper is dedicated to Hugo Theorell on the occasion of his 70th birthday. This work has been described partly in preliminary reports [1,2]. For the previous paper in this series see [3].Abbreviations. E, enzyme; R, reduced coenzyme (NADH,Enzyme. Glutamate dehydrogenase or L-glutamate:NAD(P) oxidoreductase (deaminating) (EC 1.4.1.3).Quantitative analyses of the binding of NADH to the enzyme also yielded controversial results. Some reports in the literature indicate that the coenzyme binding capacity of the enzyme depends on the state of association [ll--131, other results are contrary to that finding, indicating only one [15,16] or slightly more than one binding site per polypeptide chain and negative or positive cooperativity between these sites [l, 17,181. Circular dichroism studies give direct evidence for the existence of a second coenzy...

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Section: Discussionsupporting

confidence: 92%