Protein Science zyxwvutsrqpo (1994), 3:1493-1503. zyxwvutsrqpon Cambridge University Press. Printed in the zyxwvu USA. Copyright zyxwvutsrqp 0 1994 The Protein Society Computer modeling of substrate binding to lipases from Rhizornucor rniehei, Humicola lanuginosa, H and Candida rugosa zyxw MARTIN NORIN,' FREDRIK HAEFFNER,2 ADNANE ACHOUR,' TORBJORN NORIN,2 AND KARL HULT' ' Department of Biochemistry and Biotechnology and ' Department of Chemistry, Organic Chemistry, Royal Institute of Technology, S-100 44 Stockholm, Sweden (RECEIVED May 2, 1994; ACCEPTED June 28, 1994) 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 otherregion was hydrophilic. The aliphatic site should be a bind- ing cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are prob- ably 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 sec- ondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivitiesof these reactions. Keywords: calculation; enantioselectivity; free energy; water Lipases are enzymes that hydrolyze triacyl glycerides, thus lib- erating fatty acids. These enzymes are commercially important as constituents of washing detergents and as catalysts in indus- trial transesterifications of fats. The objective of this study was to increase our understanding of the enzyme-substrate interac- tions of the 2 homologous lipases from Rhizomucor miehei and Humicola lanuginosa by means of molecular modeling techniques. Lipases are able to catalyze a number of mutually related nucleophilic substitutions and are widely used inprepar- ative biotransformations of organic molecules. Lipase from R. miehei shows high activity in various organic solvents (Sonnet & Moore, 1988) and is therefore a useful biocatalyst in bioor- ganic synthesis. The molecular structure of the R. miehei lipase has been thoroughly investigated by means of X-ray crystallog- raphy (U. Derewenda et al., 1992; Z.S. Derewenda et al., 1992). However, our knowledge of the molecular details of its substrate binding is poor. At the time of this writing, only 1 detailed struc- ture of a lipase-inhibitor complex (Brzozowski et al., 1991; U. Derewenda et al., 1992) is currently available (Kinemage 1). In Reprint requests to: Karl Hult, Department of Biochemistry and Bio- technology, Royal Institute of Technology, S-100 zyxwvutsr 44 Stockholm, Swe- den; e-mail: kalleQbiochem.kth.se. addition, we investigated the binding regions in the active site of the lipase from Candida rugosa. This lipase is structurally dif- ferent from the 2 fungal lipases, although the dfl-hydrolase fold (Ollis etal., 1992) of all 3 lipases studied is conserved. Recently, it has been shown for the zyxw C. rugosa lipase that a tunnel, which is unique among lipases studied to this date, is involved in the binding of the scissile fatty acid chain (Grochulski et al., 1994a). The modeled binding regions of this lipase in this work are com- pared with those of the 2 fungal lipases. The active sites of the lipases were mapped by means of a method called GRID (Goodford, 1985). This method has re- cently been used to design inhibitors of influenza virus replica- tion (von Itzstein et al., 1993) and to elucidate the catalytic mechanism of phospholipase C (Byberg et al., 1992). A cubic lattice is placed in the space to be studied. At each grid point of the lattice, the interactions of a probe with the target mol- ecule are calculated by an energy function. The resulting po- tential maps are analyzed by means of a molecular graphics program to visualize interesting binding regions. The catalyticmechanisms of lipases are most probably simi- lar to those of serine proteases. Acatalytic triad, Ser-His-Asp/ Glu, is involved in an acid-base-catalyzed cleavage of ester bonds. Also important is the stabilization of a charged oxygen 1493