Bacillus subtilis chorismate mutase is partially diffusion-controlled.
The effect of viscosogens on the enzyme-catalyzed rearrangement of chorismate to prephenate has been studied. The steady-state parameters kcat and kcat/Km for the monofunctional chorismate mutase from Bacillus subtilis (BsCM) decreased significantly with increasing concentrations of glycerol, whereas the 'sluggish' BsCM mutants C75A and C75S were insensitive to changes in microviscosity. The latter results rule out extraneous interactions of the viscosogen as an explanation for the effects observed with the wild-type enzyme. Additional control experiments show that neither viscosogen-induced shifts in the pH-dependence of the enzyme-catalyzed reaction nor small perturbations of the conformational equilibrium of chorismate can account for the observed effects. Instead, BsCM appears to be limited by substrate binding and product release at low and high substrate concentrations, respectively. Analysis of the kinetic data indicates that diffusive transition states are between 30 and 40% rate-determining in these concentration regimes; the chemical step must contribute to the remaining kinetic barrier. The relatively low value of the 'on' rates for chorismate and prephenate (approximately 2 x 106 m-1.s-1) probably reflects the need for a rare conformation of the enzyme, the ligand, or both for successful binding. Interestingly, the chorismate mutase domain of the bifunctional chorismate mutase-prephenate dehydratase from Escherichia coli, which has steady-state kinetic parameters comparable to those of BsCM but has a much less accessible active site, is insensitive to changes in viscosity and the reaction it catalyses is not diffusion-controlled.
Mattei
P
P
Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, Zürich, Switzerland.
Kast
P
P
Hilvert
D
D
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002826:Chorismate Mutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014783:Viscosity
D017931:DNA Primers; D011994:Recombinant Proteins; D002826:Chorismate Mutase