Studies of Glutamate Dehydrogenase

Abstract

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...

Substrate Specificity via Ternary Complex Formation with Glutamate Dehydrogenase

“…The binding of NADPH to enzyme in Tris buffer was followed by fluorescence spectroscopy with an Aminco Bowman single beam instrument in a 1-cm square cuvette. The excitation and emission gratings were set on 357 and 456 nm respectively [12]. The data obtained were analyzed as described by Krause et al [12].…”

“…The excitation and emission gratings were set on 357 and 456 nm respectively [12]. The data obtained were analyzed as described by Krause et al [12].…”

“…The dissociation constant of NADPH to glutamate dehydrogenase appears, by comparing several different studies [3,12,15], to be strongly influenced by ionic strength, pH and temperature. For the calculation of KF in the ternary complex studies reported below, we have determined the K d for enzyme-NADPH under the experimental conditions used throughout this study.…”

Binding to antithrombin of heparin fractions with different molecular weights

“…Similar titrations of antithrombin with high-affinity heparin fractions were monitored by fluorescence (Nordenman et al, 1978). Stoicheiometries (expressed as mol of heparin bound/mol of antithrombin) and binding constants were determined by computer-fitting the data from these titrations to a theoretical equation (Krause et al, 1974;Nordenman & Bjork, 1978a). Anticoagulant activities were determined by a whole-blood clotting assay (British Pharmacopoeia, 1968), or by a spectrophotometric assay based on the ability of heparin to accelerate the reaction between purified bovine antithrombin and thrombin (Bjork & Nordenman, 1976;Laurent et al, 1978;.…”

Activation and inhibition of phosphorylase kinase by monospecific antibodies against preparatively isolated alpha, beta and gamma subunits

“…155 kDa and a 39 kDa, BDH Standards (BDH product no. 44230, BDH Poole, England) prepared from cross-linked glutamate dehydrogenase (A. T. Charteris BDH Chemicals LTD, personal communication) (monomer = 56 kDa, [39]) containing species of the following molecular masses: 56 kDa, 112 kDa, 168 kDa and 224 kDa. The unknown molecular masses of sample polypeptides were evaluated in the following way.…”