Author/Editor		Laurell, H; Contreras, JA; Castan, I; Langin, D; Holm, C
Title		Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis
Type		članek
Source		Protein Eng
Vol. and No.		Letnik 13, št. 10
Publication year		2000
Volume		str. 711-7
Language		eng
Abstract		Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B carboxylesterases. In many of these alpha/beta hydrolases, a conserved HG-dipeptide flanks the catalytic pocket. In HSL this dipeptide is followed by two additional glycine residues. Through site-directed mutagenesis, we have investigated the importance of this motif for enzyme activity. Since the presence of multiple glycine residues in a critical region could contribute to cold adaptation by providing local flexibility, we studied the effect of mutating these residues on the psychrotolerant property of HSL. Any double mutation rendered the enzyme completely inactive, without any major effect on the enzyme stability. The partially active single mutants retained the same proportion of activity at reduced temperatures as the wild-type enzyme. These results do not support a role for the HGGG motif in catalysis at low temperatures, but provide further validation of the current three-dimensional model of HSL. Rat HSL was found to be relatively more active than human HSL at low temperatures. This difference was, however, not due to the 12 amino acids which are present in the regulatory module of the rat enzyme but absent in human HSL.
Descriptors		CONSERVED SEQUENCE AMINO ACID SEQUENCE CHOLESTEROL ESTERASE COLD ENZYME STABILITY HYDROLYSIS KINETICS MODELS, MOLECULAR MORAXELLA MUTAGENESIS, SITE-DIRECTED PROTEIN STRUCTURE, TERTIARY RATS