Chorismate Mutase/Prephenate Dehydratase from Escherichia coli K12. Modification with 5,5'-Dithio-bis(2-nitrobenzoic acid)

Abstract

1 The reaction of native chorismate mutase/prephenate dehydratase with 5,5′-dithio-bis(2-nitrobenzoic acid) (Nbs2) caused a differential inactivation of the two 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 Michaelis constant for chorismate increased about 2.5-fold while the maximum rate for the mutase activity was unaffected. Reaction of an average of two more moles of sulphydryl per mole of enzyme subunit caused a further 25% inhibition of the mutase activity. 2 Circular dichroic measurements indicated that native chorismate mutase/prephenate dehydratase has 25 %α-helix and that modification of one sulphydryl group per subunit with Nbs2 caused significant changes in the secondary structure of the enzyme and in the environment of one or more tyrosine residues. 3 Prephenate is a competitive inhibitor of the mutase activity, with an inhibition constant of 0.047 ± 0.008 mM. Modification of one sulphydryl group per subunit with Nbs2 increased the value of the inhibition constant and the Michaelis constant for chorismate by the same amount. 4 At concentrations from 5 - 1000 μM 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. 5 Prephenate protected the most reactive sulphydryl group against reaction with Nbs2; phenylpyruvate had no effect. 6 It was concluded that one sulphydryl group is involved directly in the dehydratase active site. 7 The results are interpreted as favouring the theory that chorismate mutase/prephenate dehydratase has separate active sites for the two activities, although the possibility of partially overlapping sites has not been eliminated.