Simple conformation space search protocols for the evaluation of enantioselectivity of lipases.

Two computational protocols have been evaluated regarding their ability to reproduce the enthalpic part of lipase enantioselectivity by forcefield potential energy differences (deltaV#R-S). Though the shortcomings of the approach are numerous, good qualitative results have been obtained here and elsewhere. The anticipated improvement of quantitative results by use of a second protocol, which did not impose any atom movement restrictions on the total system, was realized only in part. Seemingly, results depended not only on the design of the computational procedure but also on the enzyme-substrate combination modelled. With Candida antarctica lipase B, results diverged significantly more from an estimated deltadeltaH#R-S than with Rhizomucor miehei lipase and cutinase.

Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents.

Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed.