Lipases

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The lipolytic enzymes belong to a large family of enzymes that facilitate the degradation of lipids. Lipases and phospholipases are members of this family that have been investigated extensively (Rubin and Dennis 1997). Lipolytic enzymes are water-soluble enzymes that are characterized by their ability to hydrolize aggregated lipids with a much higher velocity than the same lipid in its monomo-lecular form. This rate enhancement at lipid-water interfaces is the central theme in the study of lipolysis and distinguishes lipases from all other kinds of water-soluble enzymes, which invariably act on monomolecularly dispersed substrates.

Lipases with known three-dimensional structures span a wide range of molecular weights from 19kDa (cutinase) to 60 kDa (C. rugosa lipase). All of them, with exception of pancreatic lipases, contain only one domain that contains the a/p-hydrolase fold (Ollis et al. 1992) made by a five-stranded P sheet and two a helices. The residues of the catalytic triad (serine, histidine and acid) are located at definite positions in this fold. A specific feature of lipases, when compared with other classical serine hydrolases, is that the catalytic site is inaccessible to substrate in solution. A structural basis for this inaccessibility was originally proposed for the lipase from Rhizomucor miehei (Brady et al. 1990; Brzozowski et al. 1991) (Fig. 3.14a). These studies have shown that in the former structure, the enzyme adopts an inactive closed conformation with a surface loop from the N-terminal domain (the flap) covering the active site, and that in the latter, lipase has an active open conformation resulting from the repositioning of the flap. The inner surface of the lid, which is exposed on opening, is sufficiently hydrophobic to facilitate association of the enzyme with a lipid interface. Thus the exposure of the catalytic residues is accompanied by a marked increase in the non-polarity of the surrounding surface, providing a plausible structural basis for the interaction of the lipase with triglyceride (TG) and diglyceride (DG) substrates.

Fig. 3.14. Gallery of ribbon structures of members from the lipase family, (a) The crystal structure of Rhizomucor miehei lipase (PDB code 3TGL) showing the a/|3 hydrolase fold and the catalytic residues as sticks, (b) The Candida rugosa lipase in complex with cholesteryl linoleate (magenta) (PDB code 1CLE). (c) Crystal structure of the lipase-colipase complex (PDB code 1ETH) with the a/|3 hydrolase domain coloured in green, the C-terminal domain coloured in dark blue and the colipase cofactor coloured in cyan. The detergent mimicking the lipid at the active site is represented as sticks. In all cases the flap is coloured in orange. Figures were prepared with PyMOL

Fig. 3.14. Gallery of ribbon structures of members from the lipase family, (a) The crystal structure of Rhizomucor miehei lipase (PDB code 3TGL) showing the a/|3 hydrolase fold and the catalytic residues as sticks, (b) The Candida rugosa lipase in complex with cholesteryl linoleate (magenta) (PDB code 1CLE). (c) Crystal structure of the lipase-colipase complex (PDB code 1ETH) with the a/|3 hydrolase domain coloured in green, the C-terminal domain coloured in dark blue and the colipase cofactor coloured in cyan. The detergent mimicking the lipid at the active site is represented as sticks. In all cases the flap is coloured in orange. Figures were prepared with PyMOL

C. rugosa lipases show a substrate binding-site formed by an extensive hydrophobic pocket together with a narrow internal tunnel in which the substrate aliphatic chains are stabilized (Ghosh et al. 1995) (Fig. 3.14b). Recent studies (Mancheno et al. 2003) propose that residues situated in the hydrophobic pocket are partly responsible for the differences in substrate specificity observed between the several closely related extracellular lipases produced by the yeast Candida rugosa. Structural inspection of their hydrophobic substrate tunnel revealed two definite regions regarding their amino-acid composition, which suggests a correlation between the aromatic/aliphatic balance and the esterase/lipase character of the enzyme (Mancheno et al. 2003). Conversely, the structural variability in narrow regions exhibited in the C. rugosa lipases family may provide to this yeast the ability to metabolize a broad spectrum of substrates, justifying the presence of several closely related isoenzymes.

Pancreatic lipases exhibit a more complicated organization related to their physiological function in mammals. Crystal structures of the human pancreatic lipase have shown that the polypeptide chain is divided into two domains bearing specific functions. The N-terminal domain, which follows the a/p-hydrolase fold as other lipases, contains the catalytic triad and is responsible for triglyceride hydrolysis. The C-terminal domain displays a P-sandwich fold and is involved in binding a small protein, the colipase (molecular mass, 10 kDa), which counteract the inhibitory effect of bile salts in the intestine (Fig. 3.14c). Crystal-structure determination of the activated lipase-colipase-micelle complex (Hermoso et al. 1997), as determined using both high-resolution X-ray and low-resolution neutron diffraction techniques, revealed that the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase (Fig. 3.5b). This structure showed that the micelle- and substrate-binding sites concern different regions of the protein complex and revealed the residues involved in micelle interaction. Interestingly, in contrast with what is generally observed with membrane proteins, the protein surfaces involved in micelle binding are amphipathic without forming patches of either hydrophilic or hydrophobic amino-acid side-chains. Considering all these facts, the authors concluded that pancreatic lipase activation is not interfacial, as proposed for other lipases, but occurs in the aqueous phase and is mediated by the colipase and a bile salt micelle.

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Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

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