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 phosphotransferase system from an Escherichia coli enzyme I mutant was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. Th...

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Bibliographic Details
Published in:Biochemistry (Easton) 1996-01, Vol.35 (1), p.236-242
Main Authors: Seok, Yeong-Jae, Lee, Byeong Ryong, Gazdar, Celia, Svenson, Ingrid, Yadla, Nalini, Peterkofsky, Alan
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Binding Sites
Cloning, Molecular
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Genes, Bacterial
Glycine
Histidine
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry
Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism
Phosphotransferases (Nitrogenous Group Acceptor) - chemistry
Phosphotransferases (Nitrogenous Group Acceptor) - metabolism
Plasmids
Point Mutation
Polymerase Chain Reaction
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Restriction Mapping
Sequence Homology, Amino Acid
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Summary:The gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system from an Escherichia coli enzyme I mutant was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. The enzyme I structural gene was mutated to change glycine-338 to a variety of other amino acid residues. Fermentation tests indicated that glycine-338 could be mutated to alanine with no gross loss in phosphotransferase activity, while mutation to valine, glutamic acid, aspartic acid, arginine, histidine, or asparagine led to significant loss of activity. An expression vector for enzyme I was mutated to change glycine-338 to a variety of other amino acid residues and highly purified mutant proteins were prepared. Analysis of phosphorylation of the proteins by PEP indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine substantially decreased phosphorylation, change to histidine or arginine drastically diminished phosphorylation, and mutation to aspartic or glutamic acids abolished phosphorylation activity. Mutation at glycine-338 influences the autophosphorylation rather than the phosphoryl transfer activity of enzyme I.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi952052k