Lipases tetrahedral intermediate

Ester hydrolysis is common in biological chemistry, particularly in the digestion of dietary fats and oils. We ll save a complete discussion of the mechanistic details of fat hydrolysis until Section 29.2 but will note for now that the reaction is catalyzed by various lipase enzymes and involves two sequential nucleophilic acyl substitution reactions. The first is a trcinsesterificatiori reaction in which an alcohol gioup on the lipase adds to an ester linkage in the tat molecule to give a tetrahedral intermediate that expels alcohol and forms an acyl... 

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. 

Studies of the ability of the lipase B from Candida antarctica (CAL-B) to catalyse the enantioselective aminolysis of esters by cis- and firms-2-phenylcycloalkanamines (54 n = 1, 3, 4) have been followed up by molecular modelling approaches in order to probe the lipase-catalysed aminolysis mechanism. CAL-B possesses a typical serine-dependent triad, so it was possible, with access to an X-ray crystal structure of CAL-B, to model a series of phosphonamidates (55 n = 1, 3, 4) as analogues of the tetrahedral intermediate (TI) resulting from attack of the amine on the carbonyl of the acyl-enzyme. The results suggested as the most plausible intermediate for the CAL-B-catalysed aminolysis a zwitterionic TI resulting from the direct His-assisted attack of the amine on to a C=0 group of the acyl-enzyme.80... 

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. 

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. 

Because the lipase active site is similar to that of serine proteases, the hydrolytic mechanism may also be analogous [5]. It is suggested that the catalytic reaction begins with the formation of a noncovalent Michaelis complex between the enzyme and the substrate, which then reacts with the nucleophilic oxygen of the serine to form a covalent tettahedral ttansition state. An acyl enzyme intermediate is then formed by cleavage of the substrate ester bond and dissociation of a protonated diacylglyceride. An activated water molecule then attacks the serine ester and forms a second tetrahedral transition state. Collapse of this ttansition state results in fatty acid release and the regeneration of the enzyme. 

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