Hormone-sensitive Lipase Is Structurally Related to Acetylcholinesterase, Bile Salt-stimulated Lipase, and Several Fungal Lipases

Abstract

Hormone-sensitive lipase is the key enzyme in the mobilization of fatty acids from adipose tissue, thereby playing a crucial role in the overall energy homeostasis in mammals. Its activity is stimulated by catecholamines through cAMP-dependent phosphorylation of a single serine, a process that is prevented by insulin. This regulatory property is unique to this enzyme among all known lipases and has been acquired during evolution through insertion of a regulatory module into an ancestral lipase. Sequence alignments have failed to detect significant homology between hormone-sensitive lipase and the rest of the mammalian lipases and esterases, to which this enzyme is only very distantly related. In the present work, we report the finding of a remarkable secondary structure homology between hormone-sensitive lipase and the enzymes from a superfamily of esterases and lipases that includes acetylcholinesterase, bile salt-stimulated lipase, and several fungal lipases. This finding, based on the identification of the secondary structure elements in the hormone-sensitive lipase sequence, has allowed us to construct a three-dimensional model for the catalytic domain of hormone-sensitive lipase. The model reveals the topological organization, predicts the components of the catalytic triad, suggests a three-dimensional localization of the regulatory module, and provides a valuable tool for the future study of structural and functional aspects of this metabolically important enzyme.In mammals, free fatty acids derived from adipose tissue triglycerides are the most important fuel and provide more than half of the caloric requirements when dietary substrates are lacking. Hormone-sensitive lipase (HSL) 1 plays a crucial role in the mobilization of free fatty acids from adipose tissue by catalyzing the first and rate-limiting step in the hydrolysis of stored triglycerides. Thus, HSL is a key enzyme not only in lipid metabolism but also in overall energy homeostasis. HSL is under acute hormonal and neural control. Its activity is stimulated by cAMP-dependent phosphorylation of a single serine residue (1), named regulatory site. This phosphorylation is catalyzed by cAMP-dependent protein kinase upon stimulation of ␤-adrenergic receptors by catecholamines. The antilipolytic action of insulin is mediated through a net dephosphorylation of the regulatory site of HSL (1). A second, cAMP-independent, phosphorylation site is present in the HSL protein, two residues C-terminal of the regulatory site (1, 2). Phosphorylation of this site (called the basal site) does not affect the catalytic activity but prevents phosphorylation of the regulatory site, thus having a potential antilipolytic role (3). The regulation of the enzyme activity by reversible phosphorylation is the most remarkable property of HSL, and unique among all known lipases. A second distinctive feature of HSL is its unusual substrate specificity, hydrolyzing cholesteryl esters and triglycerides at similar rates and showing 10 times higher hydrolytic activity toward diglycer...

Identification of essential aspartic acid and histidine residues of hormone‐sensitive lipase: apparent residues of the catalytic triad

“…3). This observation was consistent in all experiments performed, and probably reflects a reduced stability of the mutant protein In order to probe the different candidate residues as to the active site aspartic acid and histidine in HSL, and to obtain experimental data to support our model for the catalytic core of this enzyme [13], residues Asp-445, Asp-703, His-493 and His-733 in rat HSL were subjected to site-directed mutagenesis. As further control residues, the conserved Asp-517 and His-696 were also mutated.…”

“…The residues that were mutated in this work are shown in ball-and-stick representation, with the exception of His-493 and Asp-517. These two residues are located outside the region modeled, in an external module located between strands ß6 and ß7 [13]. Aspartic acid residues were mutated to asparagine and/or glutamic acid and histidines to serine and/or alanine in order to minimize the disturbance of protein structure [20].…”

“…By comparing the predicted secondary structure elements with those of lipases and esterases of known structure from the esterase B family (Geotrichum candidum lipase, Candida rugosa lipase and Torpedo californica acetylcholine esterase), we have very recently built a three-dimensional model for the catalytic core of HSL [13]. One direct consequence of the structural model was the proposal of a complete catalytic triad: Ser-423 1 , Asp-703 and His-733.…”

“…HSL sequence and prediction of secondary structure elements revealed that the C-terminal domain is evolutionary related to enzymes of the esterase B family [13]. By comparing the predicted secondary structure elements with those of lipases and esterases of known structure from the esterase B family (Geotrichum candidum lipase, Candida rugosa lipase and Torpedo californica acetylcholine esterase), we have very recently built a three-dimensional model for the catalytic core of HSL [13].…”

Identification of catalytically essential residues in Escherichia coli esteraseby site‐directed mutagenesis

“…BFAE has a typical c~/[3 hydrolase fold with a unique extended N-terminal subdomain, and is composed of a eight-stranded 13-sheet and 11 (z-helices. The high resemblance of this structure to a model for the three-dimensional structure of the catalytic domain of HSL [7] strongly suggests that all proteins in the HSL family share a common structure.…”