title: Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta.
abstract: The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Brønsted plots relating the pKa of the leaving group to the observed kinetic parameters, Vmax and V/Km, are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pKa of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on Vmax and V/Km for the same series of analogues. The data were fitted to the scheme E k1A in equilibrium k2 EA k3----EP k7----E'P k9----E + products where EA is the enzyme-substrate complex, EP is the enzyme-product complex, E'P is the enzyme-product complex after a viscosity-independent unimolecular reaction, and the values for k1, k2, k7, and k9 are 4.1 X 10(7) M-1 s-1, 2550 s-1, 3370 s-1, and 5940 s-1, respectively. The magnitude of the chemical step, represented by k3, is dependent on the pKa of the leaving group phenol as predicted by the Brønsted equation (log k3 = beta pKa + C) where beta = -1.8 and the constant (C) = 17.7. The magnitude of beta indicates that the transition state for substrate hydrolysis is very product-like.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station 77843.
authors.forename: S R
authors.initials: SR
authors.lastname: Caldwell
authors.forename: J R
authors.initials: JR
authors.lastname: Newcomb
authors.forename: K A
authors.initials: KA
authors.lastname: Schlecht
authors.forename: F M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D043303:Aryldialkylphosphatase; D004058:Diffusion; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010755:Organophosphates / Q000378:metabolism*; D010261:Paraoxon / Q000031:analogs & derivatives / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000138:chemical synthesis / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D012997:Solvents / Q000378:metabolism; D013329:Structure-Activity Relationship; D014783:Viscosity
chemical_list: D010755:Organophosphates; D010636:Phenols; D011994:Recombinant Proteins; D012997:Solvents; D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase; D010261:Paraoxon