Interconvertible molecular-weight forms of the bifunctional chorismate mutase-prephenate dehydratase from Acinetobacter calcoaceticus

Abstract

Acinetobacter calcoaceticus belongs to a large phylogenetic cluster of gram-negative procaryotes that all utilize a bifunctional P-protein (chorismate mutase-prephenate dehydratase) [EC 5.4.99.5-4.2.1.51] for phenylalanine biosynthesis. These two enzyme activities from Ac. calcoaceticus were inseparable by gel-filtration or DEAE-cellulose chromatography. The molecular weight of the P-protein in the absence of effectors was 65,000. In the presence of l-tyrosine (dehydratase activator) or l-phenylalanine (inhibitor of both P-protein activities), the molecular weight increased to 122,000. Maximal activation (23-fold) of prephenate dehydratase was achieved at 0.85 m m l-tyrosine. Under these conditions, dehydratase activity exhibited a hysteretic response to increasing protein concentration. Substrate saturation curves for prephenate dehydratase were hyperbolic at l-tyrosine concentrations sufficient to give maximal activation (yielding a K m,app of 0.52 m m for prephenate), whereas at lower l-tyrosine concentrations the curves were sigmoidal. Dehydratase activity was inhibited by l-phenylalanine, and exhibited cooperative interactions for inhibitor binding. A Hill plot yielded an n′ value of 3.1. Double-reciprocal plots of substrate saturation data obtained in the presence of l-phenylalanine indicated cooperative interactions for prephenate in the presence of inhibitor. The n values obtained were 1.4 and 3.0 in the absence or presence of 0.3 m m l-phenyl-alanine, respectively. The hysteretic response of chorismate mutase activity to increasing enzyme concentration was less dramatic than that of prephenate dehydratase. A K m,app for chorismate of 0.63 m m was obtained. l-Tyrosine did not affect chorismate mutase activity, but mutase activity was inhibited both by l-phenylalanine and by prephenate. Interpretations are given about the physiological significance of the overall pattern of allosteric control of the P-protein, and the relationship between this control and the effector-induced molecular-weight transitions. The properties of the P-protein in Acinetobacter are considered within the context of the ubiquity of the P-protein within the phylogenetic cluster to which this genus belongs.