The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase

• Published in: Protein engineering 1996-06, Vol.9 (6), p.519-524
• Main Authors: Martinelle, Mats, Holmquist, Mats, Clausen, Ib Groth, Patkar, Shamkant, Svendsen, Allan, Hult, Karl
• Format: Article
• Language: English
• Subjects: Acylation; Binding Sites; Chemical Phenomena; Chemistry, Physical; emulsion; Esters - metabolism; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Glutamic Acid - chemistry; Humicola lanuginosa; hydrolase; Hydrolysis; kinetics; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Mitosporic Fungi - enzymology; Mitosporic Fungi - genetics; Models, Molecular; mutagenesis; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Substrate Specificity; Triglycerides - chemistry; Triglycerides - metabolism; Tryptophan - chemistry
• ISSN: 1741-0126; 0269-2139; 1460-213X; 1741-0134
• DOI: 10.1093/protein/9.6.519

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated 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 altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin. Trp89 was shown to be lessimportant in the hydrolysis of vinyl esters compared with ethylesters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data. It is suggested that Trp89 is important in the process of binding the acyl chain of thesubstrate into the activesite for optimal acylation reaction rate. The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate. Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiencywhen substrates with bulky acyl chains are used. In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35–70% activity of wild-type lipase. Areduction of both the acylation and deacylation reaction was consistent with the data.