Glutamate dehydrogenase from the extremely thermophilic archaebacterial isolate AN1

Abstract

An NADP-specific glutamate dehydrogenase (E.C. 1.4.1.4) constituted approximately 10% of the soluble cell protein of the extremely thermophilic archaebacterial isolate AN1. This enzyme was purified to homogeneity in high yield, using column chromatography on Reactive Red-Sepharose CL-6B and hydroxyapatite. The glutamate dehydrogenase was a tetramer of Mr 204 000. It was highly thermostable with half-life values of 12.5 h and 47 min at 90°C and 103°C, respectively. The pI was 4.0 and the pH optimum was 8.25 for both oxidative deamination and reductive amination reactions. Activity in the oxidative deamination direction was markedly enhanced by calcium, magnesium and manganese ions. Glutamate synthase activity was about 50% of the glutamate dehydrogenase activity. The thermostability and pH optimum of the glutamate synthase activity was the same as for the glutamate dehydrogenase activity. Apparent Km determinations at 40, 60, and 80°C revealed increases in apparent Km’s with increasing temperature. The kinetic mechanism of the forward and reverse reactions of the glutamate dehydrogenase was investigated using initial velocity studies and inhibition studies with products and substrate analogues. Many of the product inhibitions were of a nonlinear (parabolic) nature. The results of the experiments were consistent with a steady state random mechanism but not with an ordered mechanism or a rapid-equilibrium random mechanism. The results also indicated that there was a preference for the cofactors to bind the enzyme first (both reaction directions), and for 2-ketoglutarate to bind the enzyme second (reductive amination reaction). The true Km values (mM) for NADP⁺, glutamate, NADPH, 2-ketoglutarate and ammonia were 0.086, 2.0, 0.015, 0.71 and 10.7, respectively. The true Kᵢ (dissociation constant) values (mM) were 0.054, 1.27, 0.018, 1.79 and 8.03 for NADP⁺, glutamate, NADPH, 2-ketoglutarate and ammonia, respectively.