Oxyanion hole, lipases

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

Later, oxyanion holes were also discovered in other proteases, such as the cysteine protease papain, and in esterases and lipases, enzymes capable of esterification or ester hydrolysis. Interestingly, in these esterases, sometimes up to three hydrogen bond donors can be located within 3 A of the carbonyl oxygen atom, whereas such triple hydrogen bonding motifs have not yet been found in the proteases. 

The oxyanion hole geometry of three complexes is visuahzed in Figures 4.2. 4. Figure 4.2 displays the active site of trypsin complexed with a peptide inhibitor [41]. In Figure 4.3, the active site of chymotrypsin complexed with a neutral aldehyde adduct is displayed [43], and in Figure 4.4, cutinase (a lipase) with a covalently bound phosphate, a transition state analog is depicted [63]. 

Scheme 4.15 Examples of promiscuous enzymatic reactions conducted with the oxyanion hole of Candida antarctica lipase B (a) the aldol reaction [104] (b) the conjugate addition reaction (Michael addition) [105] (c) the epoxidation reaction [106],...

Lipases belong to the subclass of serine hydrolases, and their structure and reaction mechanism are well understood. Their common a/p-hydrolase enzyme fold is characterized by an a-helix that is connected with a sharp turn, referred to as the nucleophilic elbow, to the middle of a P-sheet array. All lipases possess an identical catalytic triad consisting of an Asp or Gin residue, a His and a nucleophilic Ser [14]. The latter residue is located at the nucleophilic elbow and is found in the middle of the highly conserved Gly—AAl—Ser—AA2—Gly sequence in which amino acids AAl and AA2 can vary. The His residue is spatially located at one side of the Ser residue, whereas at the opposite side of the Ser a negative charge can be stabilized in the so-called oxyanion hole by a series of hydrogen bond interactions. The catalytic mechanism of the class of a/P-hydrolases is briefly discussed below using CALB as a typical example, since this is the most commonly applied lipase in polymerization reactions [15]. 

In general terms, the crystallographic results show that lipases contain several distinct sites, each responsible for a specific function. The hydrolysis of the ester bond is accomplished by the catalytic triad, responsible for nucleophilic attack on the carbonyl carbon of the scissile ester bond, assisted by the oxyanion hole, which stabilizes the tetrahedral intermediates. The fatty acid recognition pocket defines the specificity of the leaving acid. There is also one or more interface activation sites, responsible for the conformational change in the enzyme. In this section the discussion is on the available structural data relevant to the function of all these sites. 

In lipases the existing database regarding the oxyanion holes is still limited. In RmL two amide groups (residues 145 and 146) were originally proposed as likely candidates for this function (Brady et al., 1990). However, structural analyses of the two RmL-inhibitor complexes (Brzozowski et al., 1991 U. Derewenda et al., 1992) revealed that the oxyanion hole is likely to be fully formed only after the conformational change associated with interfacial activation, and that it is made up of both the amide and the side-chain hydroxyl of Ser-82 (Fig. 6). A hy-... 

Figure 9.15 Carboxypeptidase IL The structure of carboxypeptidase II from wheat (right) is illustrated with its two chains (blue and red). Notice that the catalytic triad of carboxypeptidase II (left) is composed of the same amino acids as those in chymotrypsin, despite the fact that the enzymes display no structural similarity. The residues that form the oxyanion hole are highlighted in yellow. This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. All these enzymes make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. [Drawn from IWHS.pdb.]...

Summarize the roles of the catalytic triad in the mechanism of chymotrypsin and the relationship of the oxyanion hole to the tetrahedral intermediate of the reaction. Appreciate that these features are present in other proteases, esterases, and lipases. 

The mechanism of lipase-catalysed esterification or hydrolysis is shown in Scheme 4.1. The mechanism involves the formation of two tetrahedral intermediates, the first formed by nucleophilic attack of the serine residue of the catalytic triad onto the substrate. The tetrahedral intermediate loses water (R = H) or an alcohol (R H) to give an acyl enzyme complex that is either attacked by water (R = H) for hydrolysis or an alcohol (R H) for acylation. A second tetrahedral intermediate is formed that dissociates from the enzyme to give an ester or acid, thus regenerating the Hpase in its native form. Both of the tetrahedral intermediates involved in the mechanism are stabilized by hydrogen bonds to the oxyanion hole. 

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