Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis

Mammalian hormone-sensitive lipase (HSL) has given its name to a family of primarily prokaryotic proteins which are structurally related to type B car boxylesterases. In many of these alp hydrolases, a conserved HG-dipeptide h anks the catalytic pocket. In HSL this dipeptide is followed by two additio nal glycine residues. Through site-directed mutagenesis, we have investigat ed the importance of this moth for enzyme activity. Since the presence of m ultiple glycine residues in a critical region could contribute to cold adap tation by providing local flexibility, we studied the effect of mutating th ese residues on the psychrotolerant property of HSL. Any double mutation re ndered the enzyme completely inactive, without any major effect on the enzy me stability, The partially active single mutants retained the same proport ion of activity at reduced temperatures as the wild-type enzyme. These resu lts do not support a role for the HGGG motif in catalysis at low temperatur es, but provide further validation of the current three-dimensional model o f 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.