Probing a functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase through transesterification reactions in organic solvent.
To reveal the functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased the Vmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The Km,app for tributyrin was not affected by the Glu87Ala mutation, but the Km,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreased Vmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the Km,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased the Km,app for tributyrin and vinyl butyrate by a factor of > 5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0-680 mM) studied here. With vinyl butyrate as a substrate the Vmax,app was only 6% of that obtained with wild-type enzyme.
Holmquist
M
M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Clausen
I G
IG
Patkar
S
S
Svendsen
A
A
Hult
K
K
D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism; D003510:Cyclohexanes; D004951:Esterification; D005973:Glutamine; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003904:Mitosporic Fungi / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012997:Solvents; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism; D014364:Tryptophan; D014753:Vinyl Compounds / Q000378:metabolism
D002087:Butyrates; D003510:Cyclohexanes; D012997:Solvents; D014280:Triglycerides; D014753:Vinyl Compounds; D005973:Glutamine; C095498:vinyl butyrate; D014364:Tryptophan; D008049:Lipase; C005830:tributyrin