Kinetics and Regulation of the NAD(P)H-Dependent Glyoxylate-Specific Reductase from Spinach Leaves

Abstract

Abstract 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 Ki of ca. 2 mᴍ. Studies with several metabolites used as possible effectors 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 effectors 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.