IMPORTANCE OF THE REGION AROUND GLYCINE-338 FOR THE ACTIVITY OF ENZYME-I OF THE ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-SUGAR PHOSPHOTRANSFERASE SYSTEM

The gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotra nsferase system from an Escherichia call enzyme I mutant was cloned an d sequenced. The mutation was shown to be a guanine to adenine transit ion resulting in an altered protein in which glycine-338 was replaced by aspartic acid. The enzyme I structural gene was mutated to change g lycine-338 to a variety of other amino acid residues. Fermentation tes ts indicated that glycine-338 could be mutated to alanine with no gros s loss in phosphotransferase activity, while mutation to valine, gluta mic acid, aspartic acid, arginine, histidine, or asparagine led to sig nificant loss of activity. an expression vector for enzyme I was mutat ed to change glycine-338 to a variety of other amino acid residues and highly purified mutant proteins were prepared. analysis of phosphoryl ation of the proteins by PEP indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine subs tantially decreased phosphorylation, change to histidine or arginine d rastically diminished phosphorylation, and mutation to aspartic or glu tamic acids abolished phosphorylation activity. Mutation at glycine-33 8 influences the autophosphorylation rather than the phosphoryl transf er activity of enzyme I.