The binding of tyrosine and NAD+ to chorismate mutase/prephenate dehydrogenase from Escherichia coli K12 and the effects of these ligands on the activity and self-association of the enzyme. Analysis in terms of a model.

In the presence of NAD', tyrosine inhibits both activities of the bifunctional enzyme chorismate mutase/ prephenate dehydrogenase (EC 5.4.99.5/1.3.1.12).The maximal inhibition in the presence of 0.5 m~ NAD' and 0.15 l z l ~ concentration of the other substrate was 45% and 95% for the chorismate mutase and prephenate dehydrogenase activities, respectively.Binding studies using an air-driven ultracentrifuge indicated that the affinity of the enzyme for tyrosine was increased in the presence of NAD' and the affinity for NAD' was increased in the presence of tyrosine.These effects were found to be mediated by a change in the state of aggregation of the enzyme.Ultracentrifugation of the enzyme in the absence of ligands gave a single boundary and the sedimentation coefficient of the enzyme corresponded to the value expected for the dimer (szOsl, = 4.85).In the presence of 0.5 m M tyrosine or NAD', a bimodal reaction boundary was observed, while in the presence of both 0.5 m~ NAD' and tyrosine a single boundary was again observed with a sedimentation coefficient (sz0,J of 7.6 S, suggesting the formation of a tetramer.,Sedimentationequilibrium studies confirmed that these ligands increase the molecular weight of the enzyme to that of a tetramer.Other ligands such as hydroxyphenylpyruvate, NADH, and prephenate did not cause self-association.The results of these studies have been analyzed in terms of a dimer-tetramer model in which it is postulated that NAD' and tyrosine bind preferentially to the tetramer.Using the model, the inhibition of chorismate mutase activity by tyrosine in the presence of NAD' can be interpreted as resulting from a lowering of either the maximal enzyme velocity or the substrate binding affinity as the enzyme dimers associate to tetramers.The inhibition of prephenate dehydrogenase activity can also be described adequately by the model.In Escherichia coli, the first two reactions in the tyrosine biosynthetic pathway are catalyzed by the bifunctional enzyme, chorismate mutase/prephenate dehydrogenase:Chorismate --f prephenate Prephenate + NAD' -+.4-hydroxyphenylpyruvate + COz + NADH