Chemical modification of histidyl and lysyl residues in yeast enolase.
Modification of yeast enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) by diethyl pyrocarbonate at either pH 6.1 or 6.6 caused a biphasic inactivation of the enzyme. In the presence of excess Mg2+, either an equilibrium mixture of substrates or 3-phosphoglycerate, a competitive inhibitor, prevented the second slower phase of inactivation, but had no effect on the first rapid phase. Complete inactivation by diethyl pyrocarbonate correlates with the modification of six histidyl residues/subunit, while 3-phosphoglycerate protects two histidyl residues/subunit from modification. Modification of enolase by two lysine-specific reagents, 2,4,6-trinitrobenzenesulfonate and pyridoxal 5'-phosphate, at pH 8.3 caused a slow loss of enzyme activity. However, substrates did not significantly protect against inactivation by either reagent, and inactivation with 2,4,6-trinitrobenzenesulfonate correlates with the modification of 18 lysyl residues/enzyme subunit.
D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004793:Enzyme Reactivators; D006639:Histidine / Q000037:antagonists & inhibitors*; D006898:Hydroxylamines / Q000494:pharmacology; D008239:Lysine / Q000037:antagonists & inhibitors*; D009994:Osmolar Concentration; D010751:Phosphopyruvate Hydratase; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D015003:Yeasts / Q000201:enzymology*
D004793:Enzyme Reactivators; D006898:Hydroxylamines; D006639:Histidine; D011732:Pyridoxal Phosphate; D014302:Trinitrobenzenesulfonic Acid; D010751:Phosphopyruvate Hydratase; D008239:Lysine; D004047:Diethyl Pyrocarbonate