Functional Group Characterization of Homoserine Kinase fromEscherichia coli

Abstract

Homoserine kinase (EC 2.7.1.39), a key enzyme in the aspartate pathway of amino acid biosynthesis inEscherichia coli,catalyzes the phosphorylation ofl-homoserine to forml-homoserine phosphate. TheThrBgene 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 forl-homoserine shows that the enzyme loses activity upon protonation of a single functional group and upon deprotonation 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.