Substitution of Aspartate and Glutamate for Active Center Histidines in the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System Maintain Phosphotransfer Potential

Abstract

The active center histidines of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system proteins; histidine-containing protein, enzyme I, and enzyme IIAGlc were substituted with a series of amino acids (serine, threonine, tyrosine, cysteine, aspartate, and glutamate) with the potential to undergo phosphorylation. The mutants [H189E] enzyme I, [H15D]HPr, and [H90E]enzyme IIAGlc retained ability for phosphorylation as indicated by [ 32P]phosphoenolpyruvate labeling. As the active center histidines of both enzyme I and enzyme IIAGlc undergo phosphorylation of the N ε2 atom, while HPr is phosphorylated at the N δ1 atom, a pattern of successful substitution of glutamates for Nε2 phosphorylations and aspartates for N δ1 phosphorylations emerges. Furthermore, phosphotransfer between acyl residues: P-aspartyl to glutamyl and P-glutamyl to aspartyl was demonstrated with these mutant proteins and enzymes.