title: Functional group characterization of homoserine kinase from Escherichia coli.
abstract: Homoserine kinase (EC 2.7.1.39), a key enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the phosphorylation of L-homoserine to form L-homoserine phosphate. The ThrB gene coding for this enzyme has been cloned, and the enzyme has been overexpressed and purified to homogeneity with a simplified purification scheme. An examination of the pH dependence of the V/K profile for L-homoserine shows that the enzyme loses activity upon protonation of a single functional group and upon de-protonation of a second functional group, with both groups appearing to be of the cationic acid type. Incubation of the enzyme with diethylpyrocarbonate leads to the complete loss of enzyme activity. Spectral and chemical characterization of the derivatized enzyme has shown that this activity loss is caused by the modification of a histidine residue. Treatment of the enzyme with pyridoxal-5'-phosphate also results in enzyme inactivation. The spectra evidence for the formation of a Schiff base, and the complete protection afforded by substrates and inhibitors, indicate that homoserine kinase also contains a lysine that is essential for catalytic activity.
authors.affiliation: Department of Chemistry, The University of Akron, Akron, Ohio 44325-3601, USA.
authors.forename: X
authors.initials: X
authors.lastname: Huo
authors.forename: R E
authors.initials: RE
authors.lastname: Viola
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D011732:Pyridoxal Phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021370:homoserine kinase; D004047:Diethyl Pyrocarbonate