Cutinase from Fusarium solani pisi Hydrolyzing Triglyceride Analogs. Effect of Acyl Chain Length and Position in the Substrate Molecule on Activity and Enantioselectivity

• Published in: Biochemistry (Easton) 1995-05, Vol.34 (19), p.6400-6407
• Main Authors: Mannesse, Maurice L. M, Cox, Ruud C, Koops, Bart C, Verheij, Hubertus M, de Haas, Gerard H, Egmond, Maarten R, van der Hijden, Harry T. W. M, de Vlieg, Jacob
• Format: Article
• Language: English
• Subjects: Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - metabolism; Fusarium - enzymology; Fusarium solani pisi; Hydrolysis; Kinetics; Protein Structure, Tertiary; Stereoisomerism; Structure-Activity Relationship; Substrate Specificity; Triglycerides - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00019a020

Triglyceride analogues were synthesized in which one of the primary acyl ester functions has been replaced by an alkyl group and the secondary acyl ester bond has been replaced by an acyl amino bond. The chain length at either position was varied, and both (R)- and (S)-enantiomers of each compound were synthesized. These pseudo triglycerides contain only one hydrolyzable ester bond, and they are ideally suited to studying 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 distribution of the acyl chains and that the highest activities are found when the chains at positions 1 and 3 contain three or four carbon atoms. 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 enantioselectivity 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 fixed during hydrolysis. Therefore, these substrates were used to characterize mutants of cutinase with mutations in putative lipid binding domains. Two mutants (A85F and A85W) have increased activities. The results obtained with these mutants suggest an interaction of the acyl chain of the scissile ester bond with a surface loop, comprising residues 80-90, in the enzyme-substrate complex.