Chemical modification ofPseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: Kinetic evidence for an essential histidyl residue on alpha subunit

Malonyl-CoA synthetase fromPseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M^?1 min^?1 atpH 7.0, 25°C, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. The inactivated enzyme at low concentration of DEP (<0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (>0.5 mM), indicating that there may be another functional group modified by DEP. Complete inactivation of malonyl-CoA synthetase required the modification of seven residues per molecule of enzyme; however, only one is calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity.pH dependence of inactivation indicated the involvement of a residue with apK _a of 6.7, which is closely related to that of histidyl residue of proteins. Whena subunit treated with DEP was mixed with β subunits complex, the enzyme activity completely disappeared, whereas when β subunit complex treated with the reagent was mixed witha subunit, the activity remained. Inactivation of the enzyme by the reagent was protected by the presence of malonate and ATP. These results indicate that a catalytically essential histidyl residue is located at or near the malonate and ATP binding region ona subunit of the enzyme.