Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine.
The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total molecular weight 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 three unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl 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.
D000595:Amino Acid Sequence; D002498:Centrifugation; D055598:Chemical Phenomena; D002621:Chemistry; D002826:Chorismate Mutase / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007460:Iodoacetamide; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011303:Prephenate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D013820:Thermolysin; D014357:Trypsin
D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D010088:Oxidoreductases; D011303:Prephenate Dehydrogenase; D014357:Trypsin; D013820:Thermolysin; D007535:Isomerases; D002826:Chorismate Mutase; D003545:Cysteine; D007460:Iodoacetamide