Trp89 in the lid of Humicola lanuginosa lipase is important for efficient hydrolysis of tributyrin.
To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) from Humicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax = 6000 +/- 300 mmol.min-1.g-1 and an apparent Km = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Holmquist
M
M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Martinelle
M
M
Clausen
I G
IG
Patkar
S
S
Svendsen
A
A
Hult
K
K
D003902:Detergents / Q000378:metabolism; D004655:Emulsions; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009930:Organic Chemicals; D014280:Triglycerides / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D003902:Detergents; D004655:Emulsions; D009930:Organic Chemicals; D014280:Triglycerides; C089874:Triton DF 16; D014364:Tryptophan; D008049:Lipase; C005830:tributyrin