A new covalent active site reagent, l-chloro-2- oxo-6-hexanol 6-phosphate, has been synthesized from glutaric acid monomethyl ester and characterized by NMR spectroscopy. Inactivation of phosphoglucose isomerase, when incubated with various modifier concentrations, was found to be pseudo first order with respect to enzyme concentration (half-life of inactivation 6 h at pH 7.5 (30 °C) and 2.0 ì active site concentration) but showed saturation kinetics for the dependence on inactivator concentration. This saturation phenomenon demonstrates the occurrence of a reversible enzyme- inhibitor complex (Kdiss = 14.3 µM) preceding the irreversible inactivation via the chloromethyl oxo groups. Substrate or competitive inhibitors such as 6-phosphogluconate or 5-phosphoarabinonate protect against inactivation of the isomerase by the modifying reagent.
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