MODIFICATION OF MAIZE NADP-MALIC ENZYME BY WOODWARDS REAGENT-K

Maize leaf NADP-malic enzyme was rapidly inactivated by micromolar con centrations of Woodward's reagent K (WRK). The inactivation followed p seudo-first order reaction kinetics. The order of reaction with respec t to WRK was 1, suggesting that inactivation was a consequence of the modification of a single residue per active site. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group mod ification and also exhibited altered surface charge as seen from the e lution profile on ''Mono Q'' anion exchange column and the mobility on native polyacrylamide gel electrophoresis. Substrate NADP and NADP+Mg 2+ strongly protected the enzyme against WRK inactivation indicating t hat the modified residue may be located at or near the active site. Bi nding affinity of NADPH to the malic enzyme was studied by the fluores cence technique. The native enzyme binds NADPH strongly resulting in e nhancement of the fluorescence emission and also causes a blue shift i n the emission maximum of NADPH from 465 nm to 450 nm, however, the mo dified enzyme neither exhibited the enhancement of fluorescence emissi on nor the blue shift, indicating loss of NADPH binding site on modifi cation. The essential carboxyl group may be involved in NADPH binding during catalysis by the enzyme.