Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa

Abstract

The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola Ianuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme.Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.

A structural basis for enantioselective inhibition of Candida rugosa lipase by long‐chain aliphatic alcohols

“…Thus, the calculated lowest-energy conformation of the complexes is a well-defined conformation of the transition state that we believe represents the global energy minimum of the complex. This conclusion is further supported by results presented in previous reports, where the method used herein has been shown to give results that agree well with those of kinetic experiments (Norin et al, 1993(Norin et al, , 1994a). …”

“…All interactions between the substrate and the protein were thus treated in as much detail as possible to obtain the best model of the complex. In previous reports, this approach has given results that agreed well with those of kinetic experiments (Norin et al, 1993(Norin et al, , 1994a. In a region between 6 and 8 A away from the substrate, only the amino acid side chains and water molecules were allowed to move.…”

“…The modeling of the transition states followed the protocol that was used previously to calculate the enantioselectivity of chymotrypsin (Norin et al, 1993) and lipases (Norin et al, 1994a). The docking of the transition state was guided by the known crystal structure of complexes of the C. rugosa lipase with transition-state analogues (Cygler et al, 1994;Grochulski et al, 1994a).…”

“…After applying a previously developed computational method (Norin et al, 1993(Norin et al, , 1994a) involving a combination of energy minimization and molecular dynamics simulation, we found that the S-enantiomer complex had a potential energy 26 kJ/mol lower than that of the R-enantiomer complex. This result is in qualitative agreement with those of kinetic experiments with respect to the enantiopreference of the lipase.…”

“…The three-dimensional structures representing the energy minima of the diastereomeric lipase-substrate transition-state complexes were found by applying a computational protocol previously used for calculating the enantioselectivity of chymotrypsin (Norin et al, 1993) and lipases (Norin et al, 1994a). The free energy of the ground state is the same for the two enantiomeric substrates.…”