The Presence of Essential Histidine Residues in Phosphoenolpyruvate Carboxylase from Maize Leaves

A.A. IGLESIAS, C.S. ANDREO

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

Año: 1983 vol. 749 p. 9 - 17

0304-4165

Diethylpyrocarbonate inactivated maize leaf phosphoenoipyruvate carboxylase (EC 4.1.1.31) with asecond-order rate constant of 6.9 mM - i. min- 1. Activity could be restored by treatment with hydroxylamineand the pH curve of inactivation suggested the involvement of a residue having a pK a of 6.9. The ultravioletdifference spectrum of modified versus native enzyme revealed the formation of ethoxyformyl histidineresidues. Phosphoenolpyruvate, plus or minus MgCI2, protected the enzyme against inactivation, suggestingthat the modification occurred at or near the substrate-binding site. Under denaturating conditions, sevenhistidine residues per 100-kDa subunit were modified by diethylpyrocarbonate. Under non-denaturatingconditions, the carboxylase lost all of its activity after about four of the seven histidine residues per subunithad been modified by the reagent. In experiments using phosphoenolpyruvate, plus or minus MgCI2, asprotective agent, it was observed that complete inactivation resulted from the modification of only twohistidine residues per subunit. On the basis of these results it is suggested that two of the four accessiblehistidines in the native enzyme subunit are essential for substrate binding or catalysis by maize leafphosphoenolpyruvate carboxylase.