Open AccessPhospho enol pyruvate‐dependent phosphorylation site in enzyme III glc of the Escherichia coli phosphotransferase system
Author(s) -
DÖRSCHUG Michael,
FRANK Rainer,
KALBITZER Hans Robert,
HENGSTENBERG Wolfgang,
DEUTSCHER Josef
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08438.x
Subject(s) - pep group translocation , chemistry , biochemistry , bacillus subtilis , phosphoenolpyruvate carboxykinase , escherichia coli , phosphotransferase , subtilisin , enzyme , biology , bacteria , gene , genetics
Enzyme‐III glc is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimurium and is phosphorylated by phosphor enol pyruvate in a reaction requiring enzyme I (phosphor enol pyruvate‐protein phosphotransferase), and the histidine‐containing phospho‐carrier protein HPr. In this paper we report the isolation of III glc from E. coli and the characterization of the active center. Alkaline hydrolysis of [ 32 P] P ‐III glc and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P ‐III glc of S. typhimurium. Here we present 1 H‐NMR measurements of III glc and P ‐III glc from E. coli which further substantiate that the phosphoryl group in P ‐III glc is linked to the N‐3 position of a histidyl residue. After phosporylation of III glc with [ 32 P]Phospho enol pyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val‐His‐Phe‐Gly‐Ile‐Asp. The lower electrophoretic mobility of P ‐III glc on dodecylsulfate/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed‐phase column compared to unphosphorylated protein may indicate a structural change following phosphoenolpyruvate‐dependent phosphorylation.