INVESTIGADORES
GONZALEZ Daniel Hector
artículos
Título:
Active-site-directed inhibition of phosphoenolpyruvate carboxylase from maize leaves by bromopyruvate
Autor/es:
GONZALEZ, DANIEL H.; IGLESIAS, ALBERTO A.; ANDREO, CARLOS S.
Revista:
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Editorial:
ELSEVIER SCIENCE INC
Referencias:
Lugar: Amsterdam; Año: 1986 vol. 245 p. 179 - 186
ISSN:
0003-9861
Resumen:
Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase with respect to phosphoenolpyruvate (Ki:
2.3 mm 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 mm. 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 mm. 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.