ALTERED ACYL-CHAIN LENGTH SPECIFICITY OF RHIZOPUS-DELEMAR LIPASE THROUGH MUTAGENESIS AND MOLECULAR MODELING

The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specif icity for short- or medium-chain length fatty acids. Computer-generate d structural models of R. delemar lipase were used in mutant protein d esign and in the interpretation of the catalytic properties of the res ulting recombinant enzymes. Molecular dynamics simulations of the doub le mutant, va1209trp + phe112trp, predicted that the introduction of t rp112 and trp209 in the acyl binding groove would sterically hinder th e docking of fatty acids longer than butyric acid. Assayed against a m ixture of triacylglycerol substrates, the va1209trp + phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributy rin relative to the hydrolysis of tricaprylin while no triolein hydrol ysis was detected. By comparison, the val94Trp mutant, predicted to po se steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fo ld increase in tricaprylin hydrolysis relative to the hydrolysis of tr ibutyrin. Molecular models of the double mutant phe95asp + phe214arg i ndicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mix ture of triacylglycerols, the phe95asp + phe214arg substitutions resul ted in an enzyme with 3-fold enhanced relative activity for tricapryli n compared to triolein, suggesting that structural determinants for me dium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 Angstrom of the active site by the double mutation leu146lys + ser1 15asp dest royed catalytic activity entirely. Similarly, the substitution of pola r Gln at the rim of the acyl binding groove for phe112 largely elimina ted catalytic activity of the lipase.