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. 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.
Seok
Y J
YJ
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA.
Lee
B R
BR
Gazdar
C
C
Svenson
I
I
Yadla
N
N
Peterkofsky
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005998:Glycine; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D005998:Glycine