Lipase active center

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

Many excellent reviews have already been written on the subject of the catalytic centers of serine and thiol proteinases (e.g.. Kraut, 1977 Baker and Drenth, 1987 Warshel etai, 1989). In this paper the focus is specifically on the structure of the catalytic triad in lipases, with emphasis on the differences from and similarities to the catalytic centers of proteinases. The atomic coordinates for the G. candidum lipase were not available when this review was written, and the analysis of the stereochemistry of the active centers is therefore restricted to lipases from R. miehei and the human pancreas. 

The thermal stability at 60 °C of Novozyme 435, however, is higher than that of CALB-7A (Fig. 5). After 10 h of incubation at 60 °C, Novozyme 435 retained more than 70% of its initial activity, whereas CALB-7A retained only 50%. The higher stability of Novozyme 435 may be due to the hydrophobic nature of the support used for immobilization. When hydrophobic supports are used, the hydrophobic areas surrounding the enzyme active center are involved in adsorption, which stabilizes the active form of lipase [11]. 

A model for pancreatic lipase has been suggested to account for the enzyme s activity on the oil/water interface (Fig. 3.17). The lipase s hydrophobic head is bound to the oil droplet by hydrophobic interactions, while the enzyme s active site aligns with and binds to the substrate molecule. The active site resembles that of serine proteinase. The splitting of the ester bond occurs with the involvement of Ser, His and Asp residues on the enzyme by a mechanism analogous to that of chymotrypsin (cf. 2.4.2.5). The dissimilarity between pancreatic lipase and serine proteinase is in the active site lipase has a leucine residue within this site in order to establish hydrophobic contact with the lipid substrate and to align it with the activity center. 

In order to further verify the involvement of catalytic amino acid residues in enzyme-catalyzed ring-opening polymerization, catalytic amino acids of the catalytic domain of the PHB depolymerase were replaced and evaluated for their polymerization activities. PHB depolymerases have structures that consist of a catalytic domain, a putative linker region, and a substrate binding domain (SBD). Three strictly conserved amino acids, serine, aspartate, and histidine, constitute the catalytic triad at the active center of the catalytic domain. The conserved serine is part of the so-called lipase-box... 

Typically, lipases are employed in esterification however, proteases have also been utilized in organic medium although their normal function is peptidic bond hydrolysis. Their action in ester bond formation is rationalized in terms of the similarity between the active center that is mechanistically essential for transesterification. Several lipases originating from various sources have been utilized in sugar fatty acid synthesis. The activity of these enzymes is highly dependent on the reaction conditions employed and whether they are used in the free state or immobilized on a support. Immobilization confers better homogeneity of the enzyme for reaction and promotes greater thermostability of the protein [30]. The prochiral selectivity and substrate preference of lipases and protease also depend on the solvent system [34,35]. 

Among these enzymes, Novozyme 435 was the most suitable for the procedure. PS-CI and TLIM hardly catalyzed the reaction, although RMIM catalyzed it relatively well. The difference in the recognition of the substrate by lipases is probably in accordance with the difference in the substrate orientation in the active center (Pleiss et al, 1998). With one possible exception, the substrates easily accessed the active center of Novozyme 435. Additionally, it is suggested that the substrates hardly accessed the active sites of RMIM, PS-CI, and TLIM. 

Conduritols and inositols are cyclic polyalcohols with significant biological activity. The presence of four stereogenic centers in the stmcture of conduritols allows the existence of 10 stereoisomers. Enzymatic methods have been reported for the resolution of racemic mixtures or the desymmetrization of meso-conduritols. For example, Mucor miehei lipase (MML) showed enantiomeric discrimination between all-(R) and all-(S) stereoisomers ofconduritol E tetraacetate (Figure 6.52). Alcoholysis resulted in the removal of the four acetyl groups ofthe all-(R) enantiomer whereas the all-(S) enantiomer was recovered [141]. 

Enantioselective enzymatic transesterifications have been used as a complementary method to enantioselective enzymatic ester hydrolyses. The first example of this particular type of biotransformation is the synthesis of the optically active 2-acetoxy-l-silacyclohexane (5 )-78 (Scheme 19). This compound was obtained by an enantioselective transesterification of the racemic l-silacyclohexan-2-ol rac-43 with triacetin (acetate source) in isooctane, catalyzed by a crude lipase preparation from Candida cylindracea (CCL, E.C. 3.1.1.3)62. After terminating the reaction at 52% conversion (relative to total amount of substrate rac-43), the product (S)-78 was separated from the nonreacted substrate by column chromatography on silica gel and isolated in 92% yield (relative to total amount of converted rac-43) with an enantiomeric purity of 95% ee. The remaining l-silacyclohexan-2-ol (/ )-43 was obtained in 76% yield (relative to total amount of nonconverted rac-43) with an enantiomeric purity of 96% ee. Repeated recrystallization of (R)-43 led to an improvement of enantiomeric purity by up to >98% ee. Compound (R)-43 has already earlier been prepared by an enantioselective microbial reduction of the l-silacyclohexan-2-one 42 (see Scheme 8)53. The l-silacyclohexan-2-ol (R)-43 is the antipode of compound (.S j-43 which was obtained by a kinetic enzymatic resolution of the racemic 2-acetoxy-l-silacyclohexane rac-78 (see Scheme 15)62. For further enantioselective enzymatic transesterifications of racemic organosilicon substrates, with a carbon atom as the center of chirality, see References 64 and 70-72. 

Enantioselective enzymatic transesterifications have been successfully used for the synthesis of optically active silanes with the silicon atom as the center of chirality. As shown in Scheme 20, the prochiral bis(hydroxymethyl)silanes 86 and 88 were transformed into the corresponding chiral dextrorotatory isobutyrates (+)-87 and (+)-89, respectively, using Candida cylindracea lipase (CCL, E.C. 3.1.1.3) as the biocatalyst73. For these bioconversions, methyl isobutyrate was used as solvent and acylation agent. When using acetoxime isobutyrate as the acylation agent and Chromobacterium viscosum lipase (CVL ... 

All lipases of this family are characterized by a helical lid that covers the active site (Kg. 9X It has been hypothesized [145] that this lid moves away in a hydrophobic environment, thereby making the active site accessible to the lipid substrate. In this hypothesis an important role has been reserved for the bulky tryptophane residue at position 117 in the center of the helical lid. In the Rhizoptts javwicus lipase, however, this bulky hydrophobic residue is replaced by a alanine, and no other bulky residue seems located so that it could take die role of residue 117. Therefore, it was concluded that the lipid-interaction model that haa been suggested for this class of lipase is not generally applicable in all its details. 

In conclusion, the combination of an enzymatic optical resolution and subsequent chemical transformations of epimerization or racemization of the asymmetric center of the unwanted antipodes have led to the successful development of processes for preparation of the two optically active pyrethroid insecticides. This work will provide a novel feature in the application of enzymes, especially lipases for the industrial production of chiral compounds. 

Figure 9 Deduced amino acid sequences of GIDls. The two shaded amino acid residues are essential for maintaining the GA-binding activity of OsGIDI, and these are substituted by other residues in the gid1-1 or gid1-2 rice mutant arrows show the three catalytic centers (Ser (S), Asp (D), and His (H)) for hormone-sensitive lipase.

As in fungal lipases, the catalytic center is not accessible to solvent. However, instead of a simple helical lid found in RmL, or two lids seen in GcL, hPL appeared to have several loops that collectively obscure the entrance to the active site. It was subsequently observed that the displacement of two of these leads to the enzyme assuming an active conformation (van Tilbeurgh et al., 1993). One of these loops is a surface loop between residues 237 and 261, both of which are cysteines linked by a disulfide bridge. The second fragment is a shorter loop spanning residues 78 and 84. 

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