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...
Specific interaction between a-NADH and glutamate dehydrogenase is demonstrated by difference spectroscopy, circular dichroism and fluorescence measurements. Quantitative binding studies in the preparative ultracentrifuge yield six identical a-NADH binding sites per oligomer with a dissociation constant of 20 pM. Evidence for six to eight additional, very weak a-NADH binding sites is presented. Excess ADP prevents the binding of a-NADH to the tight binding sites, indicating competition of the two nucleotides. I n the enzymatic reaction, a-NADH is inactive as a coenzyme; however, like ADP, it is an activator of the reductive amination reaction of 2-oxoglutarate. a-NADH reduces the self-inhibition of high j3-NADH concentrations and thus again parallels the effects caused by ADP. Contrary to j3-NADH 01 j3-IL'ADH plus GTP, a-NADH or a-NADH plus GTP does not affect the dissociation-association equilibrium of glutamate dehydrogcnase. The results are consistent with the assumption of six binding sites for a-NADH, which are identical with the ADP binding sites and most probably the six nonactive j3-NADH binding sites.I n order to obtain more information about the coenzyme binding properties of glutamate dehydrogenase we introduced a-NADH as a coenzyme analogue. This analogue is inactive as a coenzyme in dehydrogenase reactions and has an a-glycosidic bond between the nicotinamide part and the ribose in comparison to a j3-linkage in the active coenzyme [3,4]. Preliminary experiments [l] have shown specific interactions between the enzyme and a-NADH, but we were unable to demonstrate by kinetic measurements, competition between a-NADH and the natural coenzyme, j3-NADH, for the active site of the enzyme. I n order to understand these results, we studied the interaction of a-NADH with glutamate dehydrogenase in greater detail, using spectroscopic techniques as well as direct separation methods. I n addition, steady-state kinetic measurements were carried out to elucidate the influence of a-NADH on the reaction catalyzed by glutamate dehydrogenase. MATERIALS AND METHODS Material8Beef liver glutamate dehydrogenase, a-NAD+, j3-NAD+, 8-NADH, ADP and GTP were purchased For the previous paper of this series see 121.: NAD(P) oxidoreductase (deaminating) (EC 1.4.1.3). This work has been described in a preliminary report [I]. MethodsDifference spectra were recorded with Cary 15 and 17 spectrophotometers using matched tandem cuvettes with 1-cm pathlength in each compartment. The temperature was maintained at 20 f 0.1 "C. Since difference spectroscopy is highly sensitive to concentration errors the samples were prepared by weighing all the solutions added to the cuvettes. With this procedure the concentration differences between the reference and sample cuvettes were kept below 0.05 Ole.Fluorescence spectra were measured with a Perkin-Elmer MPF-3 fluorescence spectrophotometer .The temperature was kept a t 20 f 0.1 "C by use of a jacketted cuvette holder. Circular dichroism measurements and direct binding studies of a-NADH with the...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.