Active Site Directed Inhibition of Phosphoenolpyruvate Carboxylase from Maize Leaves by Bromopyruvate

D.H. GONZÁLEZ, A.A. IGLESIAS, C.S. ANDREO

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Año: 1986 vol. 245 p. 179 - 186

0003-9861

Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylasewith respect to phosphoenolpyruvate (Ki: 2.3 mM at pH 8). Relatively low concentrationsof this compound completely and irreversibly inactivated the enzyme. The inactivationfollowed pseudo-first-order kinetics. The haloacid combines first with thecarboxylase to give a reversible enzyme-bromopyruvate complex and then alkylates theenzyme. The maximum inactivation rate constant was 0.27 min-’ at pH 7.2 and 30°Cand the concentration of bromopyruvate giving half-maximum rate of inactivation was1.8 MM. The inactivation was prevented by the substrate phosphoenolpyruvate, in theabsence or presence of MgClz, and by the competitive inhibitor P-glycolate. Malateafforded protection at pH 7 but not at pH 8. MgClz enhanced the inactivation when itwas carried out at pH 7; its effect was mainly due to a decrease in the dissociationconstant of the complex between bromopyruvate and the enzyme from 2 to 1.4 mM. Thisbehavior was not observed at pH 8. Analysis of the inactivation at different pH suggeststhat 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 enzymewith [3H]-N-ethylmaleimide suggests that the inactivation correlates with the modificationof thiol groups in the enzyme. The substrate prevented the modification of thesegroups. The results suggest that the alkylating reagent modifies cysteinyl residues atthe phosphoenolpyruvate binding site of the carboxylase.