LIMITS OF DIFFUSION IN THE HYDROLYSIS OF SUBSTRATES BY THE PHOSPHOTRIESTERASE FROM PSEUDOMONAS-DIMINUTA

The catalytic mechanism for the enzymatic hydrolysis of a series of paraoxon analogues by the phosphotriesterase from Pseudomonas diminuta has been determined. The Bronsted plots relating the pK(a) of the leaving group to the observed kinetic parameters, V(max) and V/K(m), are both nonlinear. This observation is consistent with a change in the rate-limiting step from chemical to physical events as the pK(a) of the leaving group is decreased. This conclusion is confirmed by the effects of solvent viscosity on V(max) and V/K(m) for the same series of analogues. The data were fitted to the scheme [GRAPHICS] 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 pK(a) of the leaving group phenol as predicted by the Bronsted equation (log k3 = beta-pK(a) + 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.