Chorismate mutase/prephenate dehydratase from Escherichia coli K12. Modification with 5,5'-dithio-bis (2-nitrobenzoic acid)

Gething, M.J.; Davidson, B.E.

European Journal of Biochemistry 78(1): 103-110


ISSN/ISBN: 0014-2956
PMID: 334529
DOI: 10.1111/j.1432-1033.1977.tb11718.x
Accession: 068526499

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The reaction of native chorismate mutase/prephenate dehydratase with 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2) caused a differential inactivation of the 2 enzymic activities. The dehydratase activity was completely inactivated by the reaction of 1.2 mol of sulphydryl per mol of enzyme subunit, while the mutase activity was inhibited only 5%. The Km for chorismate increased about 2.5-fold while the maximum rate for the mutase activity was unaffected. Reaction of an average of 2 more moles of sulphydryl per mole of enzyme subunit caused a further 25% inhibition of the mutase activity. Circular dichroic measurements indicated that native chorismate mutase/prephenate dehydratase had 25% .alpha.-helix and that modification of 1 sulphydryl group per subunit with Nbs2 caused significant changes in the secondary structure of the enzyme and in the environment of 1 or more tyrosine residues. Prephenate was a competitive inhibitor of the mutase activity, with an inhibition constant of 0.047 .+-. 0.008 mM. Modification of 1 sulphydryl group per subunit with Nbs2 increased the value of the inhibition constant and the Km for chorismate by the same amount. At concentrations from 5-1000 .mu.M the allosteric inhibitor phenylalanine did not markedly affect the reactivity of the most reactive sulphydryl group with Nbs2, but decreased the reactivity of the other sulphydryl groups. Prephenate protected the most reactive sulphydryl group against reaction with Nbs2; phenylpyruvate had no effect. It was concluded that 1 sulphydryl group was involved directly in the dehydratase active site. The results were interpreted as favoring the theory that chorismate mutase/prephenate dehydratase has separate active sites for the 2 activities, although the possibility of partially overlapping sites was not eliminated.