Temperature Dependence of Kinetic Parameters for Hyperthermophilic Glutamate Dehydrogenase from Aeropyrum pernix K1

Abstract

  The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50–90°C. At low concentrations of L-glutamate the K_m decreased from 2.02 to 0.56 mM and the catalytic efficiency (V_max/K_m) markedly increased (4–150 μmol•mg⁻¹•mM⁻¹) along with the increase of temperature from 50 to 90°C. At high concentrations of the substrate the K_m was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The K_m (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the K_ms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the K_ms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.