THE ROLE OF GLU87 AND TRP89 IN THE LID OF HUMICOLA-LANUGINOSA LIPASE

The importance of Glu87 and TRp89 in the lid of Humicola lanuginosa li pase for the hydrolytic activity at the water/lipid interface was inve stigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent, The Trp89 mutants displayed an alt ered chain length specificity towards triglycerides, with a higher rel ative activity towards triacetin and trioctanoin compared with tributy rin, Trp89 was shown to be less important in the hydrolysis of vinyl e sters compared with ethyl esters and triglycerides. An exclusive effec t on the acylation reaction rate by the mutation of Trp89 was consiste nt with the data, It is suggested that Trp89 is important in the proce ss of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate, The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. Th is is suggested to be due to reduction of unfavourable van der Waals c ontacts between Trp89 and the 2-substituent of the substrate, Thus, in contrast to natural substrates, Trp89 has a negative impact on the ca talytic efficiency when substrates with bulky acyl chains are used, In contrast to the Trp89 mutations, the effect on the hydrolytic activit y of the Glu87Ala mutation was almost substrate independent, 35-70% ac tivity of wild-type lipase, A reduction of both the acylation and deac ylation reaction was consistent with the data.