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

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Abstract
The NAD-specific glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase, EC 1.4.1.2) of C. sorokiniana was purified 1000-fold to electrophoretic homogeneity. The native enzyme was shown to have a MW of 180,000 and to be composed of 4 identical subunits with a MW 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 Km values for .alpha.-ketoglutarate, NADH, NH4+, NAD+ and L-glutamate were 2 mM, 0.15 mM, 40 mM, 0.15 mM and 60 mM, respectively. Whereas the Km for .alpha.-ketoglutarate and L-glutamate increased 10-fold 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the Km values for NADH and NAD+ were independent of change in pH from 7-9.6. By initial velocity, product inhibition and equilibrium substrate exchange studies, the kinetic mechanism of the 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 .alpha.-ketoglutarate and an .epsilon.-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.

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