Molecular mechanism of deactivation of C. antarctica lipase B by methanol.

The catalytic activity of Candida antarctica lipase B upon alcoholysis of a constant concentration of 15.2% vinyl acetate (vol/vol) and varying concentrations of methanol (0.7-60%) in toluene was determined experimentally by measuring the initial reaction velocity. The molecular mechanism of the deactivation of the enzyme by methanol was investigated by fitting the experimental data to a kinetic model and by molecular dynamics simulations of C. antarctica lipase B in toluene-methanol-water mixtures. The highest catalytic activity (280 U/mg) was observed at methanol concentrations as low as 0.7% methanol (vol/vol), followed by a sharp decrease at higher methanol concentrations. For methanol concentrations above 10% (vol/vol), catalytic activity was at 30% of the maximum activity. A variation of water activity in the range 0.02-0.09 had only minor effects. These experimental observations are described by a simple kinetic model using three assumptions: (1) a ping-pong bi-bi mechanism of the enzyme, (2) competitive inhibition by the substrate methanol, and (3) by describing enzyme kinetics by the thermodynamic activities of the substrates rather than by their concentrations. Two equilibrium constants of methanol (KM,MeOH=0.05 and Ki,MeOH=0.23) were derived by modeling methanol binding to the substrate binding site of the lipase in molecular dynamics simulations of protein-solvent systems at atomic resolution. Thus, the sharp maximum of catalytic activity of C. antarctica lipase B at 0.7% methanol is a direct consequence of the fact that methanol-toluene mixtures are far from ideal. Understanding the thermodynamics of solvent mixtures is prerequisite to a quantitative model of enzymatic activity in organic solvents.