Carboxamidomethylation and Inactivation of Rabbit Muscle Phosphoglucose Isomerase

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Abstract The reaction of rabbit muscle phosphoglucose isomerase with iodoacetamide was studied at both pH 5.8 and 8.55 by monitoring loss of enzymatic activity and the rate of modification of affected amino acid residues. The inactivation process was found to be of apparent first order with respect to both enzyme and iodoacetamide, at least until 90% of the original activity was lost. The inactivation was accompanied by carboxamidomethylation of histidine, cysteine, and methionine at both pH 5.8 and 8.55 and of lysine at pH 8.55 only. Substrate or the competitive inhibitor 6-phosphogluconate protected against inactivation and alkylation at either pH. At pH 5.8, 6-phosphogluconate completely prevented loss of enzymatic activity, as well as alkylation of any histidine, whereas at pH 8.55, substrate was found to afford better protection than the straight chain competitive inhibitor. The data are in agreement with the previously proposed mechanism involving the sequential action of both a lysine and a histidine residue in phosphoglucose isomerase catalysis.