title: 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 mM 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 Km,app of 0.52 mM 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 mM L-phenylalanine, respectively. The hysteretic response of chorismate mutase activity to increasing enzyme concentration was less dramatic than that of prephenate dehydratase. A Km,app for chorismate of 0.63 mM 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.
mesh_terms: D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D000494:Allosteric Regulation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D006836:Hydro-Lyases / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D010649:Phenylalanine / Q000096:biosynthesis / Q000494:pharmacology; D011302:Prephenate Dehydratase / Q000235:genetics / Q000302:isolation & purification*; D014443:Tyrosine / Q000494:pharmacology
chemical_list: D014443:Tyrosine; D010649:Phenylalanine; D006836:Hydro-Lyases; D011302:Prephenate Dehydratase