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 k inetic properties of wild type and mutated enzymes were studied with t ributyrin 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 ac tive with only 1% of the activity seen with the wild type enzyme. Ah T rp mutants had the same binding affinity to the tributyrin substrate i nterface as did the wild type enzyme. Wild type lipase showed saturati on kinetics against tributyrin when activities were measured with mixe d emulsions containing different proportions of tributyrin and the non ionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a V-max = 6000 +/- 300 mmol.min(-1).g(-1) and an apparen t K-m = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The ap parent K-m for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The act ivities of all mutants were more sensitive to the presence of Triton D F-16 in the tributyrin substrate than was wild type lipase. The activi ty of the Trp89Glu mutant was decreased to 50% in the presence of 2 vo l% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the sa me affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin i n Triton DF-16. The study showed that Trp89 located in the lid of H. l anuginosa lipase is important for the efficient hydrolysis of tributyr in and that this residue plays a role in the catalytic steps after ads orption of the lipase to the substrate interface.