L" "It "'~ ELSEVIER Biochimicaet Biophysica Acta 1289 (1996) 14-24 BR Biochi~ie~a et Biophysica A~ta NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids Maria Jose Bonete *, Francisco Perez-Pomares, Juan Ferrer, M6nica L. Camacho Departamento de Agroqufmica-BioqMmica. Facultad de Ciencias. Unicersidad de Alicante, Ap. 99. Alicante, E-03080, Spain Received 30 May 1995: revised 29 August 1995; accepted 11 September 1995 Abstract A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium. The purified halophilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity. GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme. However, the halophilic enzyme was subject to strong inhibition by TCA intermediates. When measuring the initial rate of the reaction, the oxidative deamination of L-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP +, D-glutamate and glutarate; and by dicarboxylic compounds such as adipate. On the other hand, all the amino acids tested were activators of this enzyme, except the D-isomer of the substrate L-glutamate that acted as an inhibitor. The relative effectiveness of each inhibitor or activator (K~ or K~ values) was correlated with the dipole moment (#), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity. Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors. We have also found that the best activators were amino acids with no polar lateral chain. Keywords: Archaea; Halobacterium halobium; NAD-glutamate dehydrogenase: Inhibition: Activation 1. Introduction Halophilic archaea are obligate halophiles that require 10-20% NaCI for optimal growth [1], and constitute a fascinating example of biological adaptation. The biochem- ical machinery of these organisms works at concentrations of salt at which proteins in non-halophilic cells would be salted out and cease to function [2,3]. Their proteins have a strong acidic composition that makes theirs structures salt- dependent [4,5]. The archaeon H. halobium is an extreme halophilic microorganism that needs high salt concentra- tions to grow at optimal conditions. It is chemo-organotroph and requires amino acids as organic carbon and energy sources. In its extreme environmental medium, there are relatively high concentrations of aminoacidic compounds from the increase of organic matter when water is evapo- rated. Corresponding author. Fax: +34 6 5903464: E-mail: M.J.Bonete@VM.CPD.UA.ES. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-41 65(95)00134-4 L-glutamate dehydrogenase (EC 1.4.1.2, NAD-GDH) catalyses the reversible oxidative deamination of L-gluta- mate to the corresponding keto acid, a-ketoglutarate (a key carbon intermediate), using NAD ÷ as a coenzyme: L~ -- glutamate + NAD+ + H20 - ketoglutarate + NADH + NH 4 + H + This enzyme plays an important role in the catabolism of L-glutamate [6]. Thus NAD-GDH may represent a key enzyme in the physiology of the microorganism, enabling it to convert a number of amino acids into easily utilizable metabolic intermediates, and it provides a pivotal function in linking carbon and nitrogen metabolism. It may also be important in determining the general availability of oxi- dized and reduced forms of the coenzymes [7]. A great deal is known about the enzymes of halophilic archaea. Little is known about metabolic control of the activity and synthesis of their enzymes, though they are believed to be regulated in patterns similar to enzymes of eubacteria [8]. The H. halobium uncoloured mutant strain has two different GDHs, one NADP-dependent that has a