Serine hydrolase reaction mechanism

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. 

The mechanism of serine (3-lactamases is similar to that of a general serine hydrolase. Figure 8.14 illustrates the reaction of a serine (3-lac(amasc with another type of (3-lactam antibiotic, a cephalosporin. The active-site serine functions as an attacking nucleophile, forming a covalent bond between the serine side chain oxygen... 

The previous chapter offered a broad overview of peptidases and esterases in terms of their classification, localization, and some physiological roles. Mention was made of the classification of hydrolases based on a characteristic functionality in their catalytic site, namely serine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallopeptidases. What was left for the present chapter, however, is a detailed presentation of their catalytic site and mechanisms. As such, this chapter serves as a logical link between the preceding overview and the following chapters, whose focus is on metabolic reactions. 

These three catalytic functionalities are similar in practically all hydrolytic enzymes, but the actual functional groups performing the reactions differ among hydrolases. Based on the structures of their catalytic sites, hydrolases can be divided into five classes, namely serine hydrolases, threonine hydrolases, cysteine hydrolases, aspartic hydrolases, and metallohydrolases, to which the similarly acting calcium-dependent hydrolases can be added. Hydrolases of yet unknown catalytic mechanism also exist. 

The transesterification of cocaine to cocaethylene is an enzymatic reaction catalyzed by microsomal carboxylesterases and blocked by inhibitors of serine hydrolases [124][125], In Chapt. 3, we have discussed the mechanism of serine hydrolases, showing how a H20 molecule enters the catalytic cycle to hydrolyze the acylated serine residue in the active site of the enzyme. In the case of cocaine, the acyl group is the benzoylecgoninyl moiety (Fig. 7.9,d ), which undergoes esterification with ethanol according to Steps e and/ (Fig. 7.9). 

However, not included in the above mechanisms are other amino acid side-chains at the active site, whose special role will be to help bind the reagents in the required conformation for the reaction to occur. Examples of such interactions are found with acetylcholinesterase and chymotrypsin, representatives of a group of hydrolytic enzymes termed serine hydrolases, in that a specific serine amino acid residue is crucial for the mechanism of action. 

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]. 

Lipases belong to the subclass of a/P-hydrolases and their structure and reaction mechanism are well understood. All lipases possess an identical catalytic triad consisting of an aspartate or glutamate, a histidine, and a nucleophilic serine residue [67], The reaction mechanism of CALB is briefly discussed as a typical example of lipase catalysis (Scheme 7). 

Both AChE and BChE are of the serine hydrolase class, which includes proteases such as trypsin (see PROTEASE inhibitors). Characteristically, such enzymes can be inhibited through covalent linkage of constituent parts of irreversible anticholinesterases such as dyflos (DFP, diisopropylfluorophosphonate). The active site of the enzyme contains a catalytic triad with a glutamate residue, a serine residue and a histidine imidazole ring. The mechanism of the catalysis of break down of AChE has been characterized, and the reaction progresses at a very fast rate. 

A pantothenic acid hydrolase (pantothenase) activity has been isolated from Pseudomonas fluorescens and other Pseudomonas strains. This enzyme hydrolyzes the amide bond of pantothenic acid 2 to form pantoic acid 5 (or pantoyl lactone) and /i-alanine 7 (EC 3.5.1.22) (Equation (10)). A detailed kinetic study of the reaction mechanism has shown that the reaction is partially reversible because of the formation of an acyl—enzyme (pantoyl-enzyme) intermediate during the course of catalysis, which may react with either water or / -alanine to form pantoic acid (the product hydrolysis) or pantothenic acid (the original substrate) Such a mechanism suggests that this enzyme could act as a pantothenate synthase, as reaction of the active site serine with pantoyl lactone would result in the formation of the pantoyl—enzyme intermediate. However, no biochemical or genetic evidence is currently available to support such a hypothesis. 

The protein is a 12-stranded anti-parallel p-barrel with amphipathic P-strands traversing the membrane (Fig. 4). The active-site catalytic residues are similar to a classical serine hydrolase triad except that in addition to the serine (Ser-144) and histidine (His-142), there is an asparagine (Asn-156) in place of the expected aspartic acid. Calcium at the active site is predicted to be involved in the reaction mechanism facilitating hydrolysis of the ester. 

Mechanisms of Serine Hydrolases. Typical to enzymatic reactions, the enzyme (E) first binds its substrate (S) at the active site as an enzyme-substrate complex (E S). For the formation of the product P, the enzyme-catalyzed reaction then takes place through the mechanism typical of the enzyme. At the active site of serine hydrolases (lipases, carboxylesterases, and serine proteases), the catalytic machinery is called a cataljdic triad consisting of amino acid residues Ser, His, and either Asp or Glu (Fig. 5). In the E S complex, imidazole of His serves as a general acid/base catalyst, catalyzing the addition of the alcoholic hydroxyl of the serine residue to the carbonyl carbon of the acyl donor (R C02R, the first substrate S ). This leads both to the liberation of the first product P (R OH) and to the formation of the so-called acyl-enzyme intermediate. This ester intermediate then reacts with the second substrate (R OH), which leads to the... 

Fig. 5. Top view for the ping-pong bi-bi mechanism of serine hydrolases in the reaction RICO2R2 -f R3QH R2QH -1- RICO2R3.

Hydrolases possess a common feature for its mechanistic action. Their active site is built around three key amino acid residues, which are histidine (His), serine (Ser), and aspartic acid (Asp) or alternatively glutamic acid (Glu). This site, namely, the catalytic triad, forms a charge-relay network to polarize and activate the nucleophile, allowing the serine hydrolase mechanism to start [19]. This review covers the acylation of alcohols and amines using mainly esters so Figure 9.1 depicts the mechanism for these types of reactions is depicted. 

Lipases are serine hydrolases and follow a bi-bi ping-pong reaction mechanism [2,3]. During the catalysis (Scheme 1) an acyl enzyme is formed in the acylation step where the serine hydroxyl is acylated by the acyl donor (the first substrate, RvCOOR2) [4]. The first product (HOR2) is then released. The acyl acceptor (the second substrate, HOR3), which in the natural reaction is water but can in principle by any nucleophile, then reacts with the acyl enzyme in the deacylation step to form the second product (RjCOORs) and die free enzyme. 

Sch6IT161 Reaction mechanism of a serine hydrolase. The serine hydroxyl in the enzyme active site is shown. 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Hydrolysis of epoxides, esters, amides, and related structures is an important biotransformation reaction that limits the therapeutic activity of many drugs and generates therapeutically active drugs from prodmg structures. In a few cases, hydrolytic reactions can generate a toxic structure. Epoxide hydrolases and esterases are members of the a/(3 hydrolase-fold family of enzymes (Morisseau and Hammock, 2005 Satoh and Hosokawa, 2006). Although their substrate specificities are radically different (e.g., lipids, peptides, epoxides, esters, amides, haloalkanes), their catalytic mechanisms are similar. All of these enzymes have an active site catalytic triad composed of a nucleophilic serine or cysteine residue (esterases/amidases), or aspartate residue (epoxide hydrolases) to activate the substrate, and histidine residue and glutamate or aspartate residues that act cooperatively in an acid—base reaction to activate a water molecule for the hydrolytic step. 

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