nitrophenols and 4-nitrophenyl-propionate

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (

Topics: Binding Sites; Biocatalysis; Crystallization; Esterases; Hydrolysis; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Nitrophenols; Phenylpropionates; Protein Binding; Proton Magnetic Resonance Spectroscopy; Serum Albumin, Bovine; Serum Albumin, Human

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1.s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1.s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

Topics: Acylation; Catalysis; Enterobacteriaceae; Hydrolysis; Kinetics; Mutation; Nitrophenols; Penicillin Amidase; Penicillin G; Phenylacetates; Phenylpropionates; Spectrophotometry