A STUDY ON THE INACTIVATION OF MAIZE LEAVES NADP-MALIC ENZYME BY 3-BROMOPYRUVATE

Bromopyruvate is a noncompetitive inhibitor of maize leaf NADP+-malic enzyme with respect to L-malate (K(i) 1 mM at pH 8.0). Relatively low concentrations of this compound completely and irreversibly inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The halocid combines first with the enzyme to give a reversible enzyme-bromopyruvate complex and then alkylates the enzyme. The maximum inactivation rate constant (k2) was 0.078 min-1 and the dissociation constant of the enzyme-bromopyruvate complex (K(i)) was 4.2 mM at pH 8.0. At pH 7.0 or 8.0, the presence of high Mg+2 concentrations accelerates the inactivation. The effect was mainly due to a decrease in the K(i) value from 4.2 mM without Mg+2 to 1.3 mM in the presence of 10 mM Mg+2 at pH 8.0 while k2 remained essentially unchanged. In contrast, low Mg2+ concentrations partially prevented the inactivation at pH 7.0 or 8.0. This dual behaviour of Mg2+ reflects the presence of two Mg2+-binding sites with different affinities. The inactivation was totally prevented by NADP+ at pH 7.0 or 8.0 suggesting that the enzyme-NADP+ complex cannot react with bromopyruvate. Analysis of the inactivation at different pH indicates that a group with a pK(a) near 8.0 must be unprotonated for the alkylation step. In addition, determination of the number of sulphydryl groups of the native and modified enzyme with 5,5-dithiobis(2-nitrobenzoic acid) showed that the inactivation occurred concomitant with the modification of two cysteinyl residues per subunit. The results suggest that bromopyruvate reacts with amino acid residues located at the active site (NADP+ site) of malic enzyme from maize leaves.