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Articles

Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana ¹

Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (L-glutamate:NAD⁺ oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K_m values for -ketoglutarate, NADH, NH₄⁺, NAD⁺, and L-glutamate were 2 mM, 0.15 mM, 40 mM, 0.15 mM, and 60 mM, respectively. Whereas the K_m for -ketoglutarate and L-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K_m values for NADH and NAD⁺ were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between -ketoglutarate and an -amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH₄⁺-inducible glutamate dehydrogenase in this organism.

³ Present address: Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115.

⁴ To whom reprint requests should be sent.

¹ This study was supported by Grant BMS-75-02287 from the National Science Foundation and in part by Public Health Service Grant GM19871 from the National Institute of General Medical Sciences, and is part of the thesis of M. J. M. submitted in partial fulfillment of requirements for Ph.D. degree.