Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity.
The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.
D000150:Acinetobacter / Q000201:enzymology*; D002850:Chromatography, Gel; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006836:Hydro-Lyases / Q000302:isolation & purification*; D007158:Immunologic Techniques; D007700:Kinetics; D008970:Molecular Weight; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D011302:Prephenate Dehydratase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D010649:Phenylalanine; D006836:Hydro-Lyases; D011302:Prephenate Dehydratase