KINETICS AND REGULATION OF THE NAD(P)H-DEPENDENT GLYOXYLATE-SPECIFIC REDUCTASE FROM SPINACH LEAVES

Kinetic mechanism of purified spinach leaf NAD(P)H glyoxylate reductas e (GR-1) was studied using either NADPH and NADH as alternative substr ates with glyoxylate. The mechanism was elucidated from substrate kine tic patterns using NADH as a cofactor rather than NADPH. With NADPH va ried versus glyoxylate, and with NADPH and glyoxylate varied at a cons tant 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 w ith glycolate and NADP have suggested either that NADPH binds to the e nzyme 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 li ttle 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 consisten t with the proposed role of the enzyme in detoxifying glyoxylate which may act as a potent inhibitor of photosynthetic processes in plant ti ssues.