Modification of maize NADP-malic enzyme by Woodward's reagent 'K'.
Maize leaf NADP-malic enzyme was rapidly inactivated by micromolar concentrations of Woodward's reagent K (WRK). The inactivation followed pseudo-first order reaction kinetics. The order of reaction with respect 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 modification and also exhibited altered surface charge as seen from the elution profile on "Mono Q" anion exchange column and the mobility on native polyacrylamide gel electrophoresis. Substrate NADP and NADP + Mg2+ strongly protected the enzyme against WRK inactivation indicating that the modified residue may be located at or near the active site. Binding affinity of NADPH to the malic enzyme was studied by the fluorescence technique. The native enzyme binds NADPH strongly resulting in enhancement of the fluorescence emission and also causes a blue shift in the emission maximum of NADPH from 465 nm to 450 nm, however, the modified enzyme neither exhibited the enhancement of fluorescence emission nor the blue shift, indicating loss of NADPH binding site on modification. The essential carboxyl group may be involved in NADPH binding during catalysis by the enzyme.
Rao
S R
SR
Molecular Biology and Agriculture, Division Bhabha Atomic Research Centre Trombay, Bombay, India.
Kamath
B G
BG
Bhagwat
A S
AS
D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007555:Isoxazoles / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D008293:Malates / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology
D004791:Enzyme Inhibitors; D007555:Isoxazoles; D008293:Malates; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D009249:NADP; C030298:malic acid; D008291:Malate Dehydrogenase; D008274:Magnesium