A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

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Abstract

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the Ka for chorismate and the Kis for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.

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