Monofunctional chorismate mutase from Serratia rubidaea: a paradigm system for the three-isozyme gene family of enteric bacteria.
Serratia rubidaea (ATCC 27614) typifies a substantial number of enteric bacteria which, unlike Escherichia coli, possess a monofunctional species of chorismate mutase (denoted CM-F). CM-F coexists with two additional species of chorismate mutase, each of the latter being one catalytic domain of a bifunctional protein. The two bifunctional proteins are utilized for phenylalanine (CM-P/prephenate dehydratase) and tyrosine (CM-T/cyclohexadienyl dehydrogenase) biosynthesis in all enteric bacteria. S. rubidaea was selected as the organism of choice for purification of CM-F because of the relatively abundant level of expression found for this enzyme. The monofunctional CM-F enzyme was purified about 1600-fold with a yield of about 16%. This is the first monofunctional chorismate mutase to be purified from any gram-negative prokaryote. The CM-F enzyme is a positively charged homodimer made up of 20-kDa subunits. It has a pH optimum of 5.5, exhibits a Km value of 0.33 mM for chorismate, and is sensitive to product inhibition by prephenate that is competitive with respect to chorismate. It is insensitive to feedback inhibition by any of the aromatic amino acids. Partial purification of the bifunctional P-protein and the bifunctional T-protein was also carried out in order to compare the properties of CM-F, CM-P, and CM-T in a common organism. The most striking differential properties of the three isozymes were those of pH optimum and degree of protection conferred by dithiothreitol.
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Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Jensen
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D001667:Binding, Competitive; D002826:Chorismate Mutase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D053138:Cyclohexenes; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D010649:Phenylalanine / Q000494:pharmacology; D012705:Serratia / Q000201:enzymology*
D003509:Cyclohexanecarboxylic Acids; D053138:Cyclohexenes; D007527:Isoenzymes; D046911:Macromolecular Substances; D010649:Phenylalanine; D002826:Chorismate Mutase; D004229:Dithiothreitol; C005550:prephenic acid