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

tion of the homology between HSL and the Moraxella lipase 4To whom correspondence should be addressed. E-mail: prompted us to investigate the cold adaptation of HSL and cecilia.holm@medkem.lu.se compare it with two other mammalian lipases, carboxylester Mammalian hormone-sensitive lipase (HSL) has given its lipase and lipoprotein lipase. At 10°C HSL was found to retain name to a family of primarily prokaryotic proteins which 3–5-fold more of its 37°C catalytic activity than both of these are structurally related to type B carboxylesterases. In lipases (Langin et al., 1993). This psychrotolerant property is many of these α/β hydrolases, a conserved HG-dipeptide probably of substantial survival value for obligatory hibernaflanks the catalytic pocket. In HSL this dipeptide is followed tors, whose body temperature can decrease to less than 5°C by two additional glycine residues. Through site-directed during hibernation. These animals are dependent on a coldmutagenesis, we have investigated the importance of this adapted HSL for the mobilization of fatty acids from endogenmotif for enzyme activity. Since the presence of multiple ously stored triacylglycerols in white adipose tissue (WAT) glycine residues in a critical region could contribute to cold and brown adipose tissue (BAT). Fatty acids are the major adaptation by providing local flexibility, we studied the fuel during the hibernation period and are also needed for the effect of mutating these residues on the psychrotolerant activation of uncoupling protein (UCP) in the process of nonproperty of HSL. Any double mutation rendered the shivering thermogenesis, which allows the animals to return enzyme completely inactive, without any major effect on quickly to a euthermic state. the enzyme stability. The partially active single mutants The structural basis for cold adaptation of enzymes is not retained the same proportion of activity at reduced temperknown. It has generally been assumed that cold adaptation atures as the wild-type enzyme. These results do not support or psychrophilicity is accompanied by increased flexibility, a role for the HGGG motif in catalysis at low temperatures, whereas thermophilic enzymes are characterized by a high but provide further validation of the current three-dimendegree of rigidity. Experimental evidence supporting the gensional model of HSL. Rat HSL was found to be relatively eral validity of these hypotheses is, however, lacking. The more active than human HSL at low temperatures. This crystal structure of only five psychrophilic enzymes have been difference was, however, not due to the 12 amino acids solved to date, α-amylase (Aghajari et al., 1998), a Ca2 –Zn2 which are present in the regulatory module of the rat protease (Villeret et al., 1997), triose–phosphate isomerase enzyme but absent in human HSL. (Alvarez et al., 1998), citrate synthase (Russell et al., 1998)