Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine

Abstract

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, was isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total MW 78,000 and displays Michaelis-Menten kinetics for both activities. Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-[14C]carboxymethylated enzyme allowed the identification of 3 unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5''-dithiobis(2-nitrobenzoate) or iodoacetamide showed that 1 SH group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities. This work supports previous proposals for a close spatial relationship between the active sites.