Kinetics and Regulation of the Nad(P)H-Dependent Glyoxylate-Specific Reductase from Spinach Leaves
Zeitschrift Fur Naturforschung C-a Journal of Biosciences: 1995 50:21-28
Kinetic mechanism of purified spinach leaf NAD(P)H glyoxylate reductase (GR-1) was studied using either NADPH and NADH as alternative substrates with glyoxylate. The mechanism was elucidated from substrate kinetic patterns using NADH as a cofactor rather than NADPH. With NADPH varied versus glyoxylate, and with NADPH and glyoxylate varied at a constant ratio, the patterns obtained on double reciprocal plots appeared to be consistent with a ping-pong mechanism, however, kinetic patterns with NADH conclusively ruled out the ping-pong reaction in favour of the sequential addition of the reactants. Product inhibition studies with glycolate and NADP have suggested either that NADPH binds to the enzyme before glyoxylate or that the addition of substrates is a random one. Studies with active group modifiers suggested an involvement of histidine, serine and cysteine residues in GR-1 activity. Salts had little or no effect on the activity of the enzyme, with the exception of cyanide, which had an apparent K-i of ca. 2 mM. Studies with several metabolites used as possible effecters of GR-1 activity have suggested that the enzyme is modulated only by substrate availability in vivo. The apparent insensitivity of GR-1 to metabolic effecters is consistent with the proposed role of the enzyme in detoxifying glyoxylate which may act as a potent inhibitor of photosynthetic processes in plant tissues.
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