Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagent K.
Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagent K (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP+Mg2+ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.
Maralihalli
G B
GB
Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Trombay, Bombay, India.
Bhagwat
A S
AS
D007202:Indicators and Reagents; D007555:Isoxazoles; D007700:Kinetics; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D003313:Zea mays / Q000201:enzymology*
D007202:Indicators and Reagents; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D010730:Phosphoenolpyruvate Carboxylase