CUTINASE FROM FUSARIUM-SOLANI PISI HYDROLYZING TRIGLYCERIDE ANALOGS -EFFECT OF ACYL-CHAIN LENGTH AND POSITION IN THE SUBSTRATE MOLECULE ONACTIVITY AND ENANTIOSELECTIVITY

Triglyceride analogues were synthesized in which one of the primary ac yl ester functions has been replaced by an alkyl group and the seconda ry acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomer s of each compound were synthesized. These pseudo triglycerides contai n only one hydrolyzable eater bond, and they are ideally suited to stu dying the influence of the chain length at the 1-, 2-, and 3-position on lipase activity and on stereopreference. These substrates were used to characterize cutinase from Fusarium solani pisi. Our results show that the activity of cutinase is very sensitive to the length and dist ribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atom s. The enzyme preferentially hydrolyzes the (R)-enantiomers, but this preference is strongly dependent on the acyl chain length distribution , with (R) over (S) activity ratios varying from about 30 to 1. This e nantioselectivity was found in three different assay systems: a mixed micellar, a reverse micellar, and a monolayer study, Our data suggest that at least two alkyl chains of the pseudo triglycerides must be fix ed during hydrolysis. Therefore, these substrates were used to charact erize mutants of cutinase with mutations in putative lipid binding dom ains. Two mutants (A85F and A85W) have increased activities. The resul ts obtained with these mutants suggest an interaction of the acyl chai n of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.