Chemical modification of Pseudomonas fluorescens malonyl-CoA synthetase by diethylpyrocarbonate: kinetic evidence for an essential histidyl residue on alpha subunit.
Malonyl-CoA synthetase from Pseudomonas fluorescens was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of 775 M-1 min-1 at pH 7.0, 25 degrees 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 (less than 0.2 mM) could be completely reactivated by hydroxylamine but not completely reactivated at high concentration (greater than 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 a pK alpha of 6.7, which is closely related to that of histidyl residue of proteins. When alpha subunit treated with DEP was mixed with beta subunits complex, the enzyme activity completely disappeared, whereas when beta subunit complex treated with the reagent was mixed with alpha 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 on alpha subunit of the enzyme.
Kim
Y S
YS
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
Kim
Y I
YI
Bang
S K
SK
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000737:chemistry*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D004793:Enzyme Reactivators / Q000737:chemistry*; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D008314:Malonates / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D001426:Bacterial Proteins; D004793:Enzyme Reactivators; D006898:Hydroxylamines; D046911:Macromolecular Substances; D008314:Malonates; C072750:N-carbethoxyhistidine; D019811:Hydroxylamine; D006639:Histidine; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase; D004047:Diethyl Pyrocarbonate; D003065:Coenzyme A