Archives of Biochemistry and Biophysics Volume 245, Issue 1 , 15 February 1986, Pages 179-186 Active-site-directed inhibition of phosphoenolpyruvate carboxylase from maize leaves by bromopyruvate*1 Daniel H. Gonzalez2, Alberto A. Iglesias2 and Carlos S. Andreo ,3 Centro de Estudios Fotosintéticos y Bioquímicos, Suipacha 531, 2000, Rosario, Argentina Received 1 July 1985; Revised 9 October 1985. Available online 2 November 2004. Abstract Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase with respect to phosphoenolpyruvate (Ki: 2.3 m at pH 8). Relatively low concentrations of this compound completely and irreversibly inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The haloacid combines first with the carboxylase to give a reversible enzyme- bromopyruvate complex and then alkylates the enzyme. The maximum inactivation rate constant was 0.27 min−1 at pH 7.2 and 30 °C and the concentration of bromopyruvate giving half-maximum rate of inactivation was 1.8 m . The inactivation was prevented by the substrate phosphoenolpyruvate, in the absence or presence of MgCl2, and by the competitive inhibitor P- glycolate. Malate afforded protection at pH 7 but not at pH 8. MgCl2 enhanced the inactivation when it was carried out at pH 7; its effect was mainly due to a decrease in the dissociation constant of the complex between bromopyruvate and the enzyme from 2 to 1.4 m . This behavior was not observed at pH 8. Analysis of the inactivation at different pH suggests that a group of pKa near 7.5 is important for the binding of the reagent to the carboxylase. Determination of the number of sulfhydryl groups of the native and modified enzyme with [3H]-N- ethylmaleimide suggests that the inactivation correlates with the modification of thiol groups in the enzyme. The substrate prevented the modification of these groups. The results suggest that the alkylating reagent modifies cysteinyl residues at the phosphoenolpyruvate binding site of the carboxylase.
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