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Archives of Biochemistry and Biophysics

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					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.