Nucleophilic serine residue

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). 

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). 

Serine peptidases the catalytic apparatus includes a nucleophilic serine residue. 

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

More than a third of all known proteolytic enzymes are serine proteases (2). The family name stems from the nucleophilic serine residue within the active site, which attacks the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate. Nucleophilicity of the catalytic serine is commonly dependent on a catalytic triad of aspartic acid, histidine, and serine—commonly referred to as a charge relay system (3). First observed by Blow over 30 years ago in the structure of chymotrypsin (4), the same combination has been found in four other three-dimensional protein folds that catalyze hydrolysis of peptide bonds. Examples of these folds are observed in trypsin. 

Acetylcholine is a neurotransmitter that relays nerve impulses across the neuromuscular Junction. Acetylcholinesterase (AcChE) rapidly breaks dovm acetylcholine, thereby loweringits concentration in the synaptic cleft and ensuring that nerve impulses are of a finite length. As shown in Fig. 17.38, a nucleophilic serine residue reacts with the substrate to form an acetyl-serine intermediate (100) with concomitant release of choline. This intermediate is then rapidly hydrolyzed by wa-... 

Proposals made concerning this hydrolysis mechanism suggest that the active site of AChE consists of a nucleophilic serine residue and a histidine residue which probably serves as a proton donor or receiver (6). Hydrolysis is thought to occur through the formation of a Michealis complex between the active site of the AChE and the substrate (S), the conversion of the complex in an "acylation" reaction to an acyl enzyme intermediate (AChE-A) and the product choline (or thiocholine), and the subsequent hydrolysis of the acyl enzyme in a "deacylation" reaction to acetic acid and the regenerated enzyme (1,8,9). 

The presence of a nucleophilic serine residue means that water is not required in the initial stages of the mechanism. This is important since water is a poor nucleophile and may also find it difficult to penetrate a hydrophobic active site. Secondly, a water molecule would have to drift into the active site, and search out the carboxyl group before it could attack it. This would be something similar to a game of blind man s buff. The enzyme, on the other hand, can provide a serine OH group, positioned in exactly the right spot to react with the substrate. Therefore, the nucleophile has no need to search for its substrate. The substrate has been delivered to it. 

The reaction between esterase and phosphorus inhibitor (109) is bimolecular, of the weU-known S 2 type, and represents the attack of a nucleophilic serine hydroxyl with a neighboring imida2ole ring of a histidine residue at the active site, on the electrophilic phosphorus atom, and mimics the normal three-step reaction that takes place between enzyme and substrate (reaction ). 

The metabolic breakdown of triacylglycerols begins with their hydrolysis to yield glycerol plus fatty acids. The reaction is catalyzed by a lipase, whose mechanism of action is shown in Figure 29.2. The active site of the enzyme contains a catalytic triad of aspartic acid, histidine, and serine residues, which act cooperatively to provide the necessary acid and base catalysis for the individual steps. Hydrolysis is accomplished by two sequential nucleophilic acyl substitution reactions, one that covalently binds an acyl group to the side chain -OH of a serine residue on the enzyme and a second that frees the fatty acid from the enzyme. 

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 antibiotic activity of certain (3-lactams depends largely on their interaction with two different groups of bacterial enzymes. (3-Lactams, like the penicillins and cephalosporins, inhibit the DD-peptidases/transpeptidases that are responsible for the final step of bacterial cell wall biosynthesis.63 Unfortunately, they are themselves destroyed by the [3-lactamases,64 which thereby provide much of the resistance to these antibiotics. Class A, C, and D [3-lactamases and DD-peptidases all have a conserved serine residue in the active site whose hydroxyl group is the primary nucleophile that attacks the substrate carbonyl. Catalysis in both cases involves a double-displacement reaction with the transient formation of an acyl-enzyme intermediate. The major distinction between [3-lactamases and their evolutionary parents the DD-peptidase residues is the lifetime of the acyl-enzyme it is short in (3-lactamases and long in the DD-peptidases.65-67... 

Catalytic site of lipase is known to be a serine-residue and lipase-catalyzed reactions are considered to proceed via an acyl-enzyme intermediate. The mechanism of lipase-catalyzed polymerization of divinyl ester and glycol is proposed as follows (Fig. 3). First, the hydroxy group of the serine residue nucleophilically attacks the acyl-carbon of the divinyl ester monomer to produce an acyl-enzyme intermediate involving elimination of acetaldehyde. The reaction of the intermediate with the glycol produces 1 1 adduct of both... 

In a lipase-catalyzed reaction, the acyl group of the ester is transferred to the hydroxyl group of the serine residue to form the acylated enzyme. The acyl group is then transferred to an external nucleophile with the return of the enzyme to its preacylated state to restart the catalytic cycle. A variety of nucleophiles can participate in this process. For example, reaction in the presence of water results in hydrolysis, reaction in alcohol results in esterification or transesterification, and reaction in amine results in amination. Kirchner et al.3 reported that it was possible to use hydrolytic enzymes under conditions of limited moisture to catalyze the formation of esters, and this is now becoming very popular for the resolution of alcohols.4... 

This example illustrates that the reactivity of peptide bonds involving serine residues is not due solely to the presence of serine other, as yet poorly understood factors must also play a role. Threonine residues also show this particular type of reactivity [9]. Here, we examine the case of cyclosporin A (CsA), a cyclic undecapeptide that contains some unusual amino acids (6.57, Fig. 6.23), whose major breakdown reaction is hydrolytic cleavage at a threonine analogue. Position 1 is occupied by 4-[( )-but-2-enyl]-A,4-dimethyl-threonine, whose OH group reacts as a nucleophile at the carbonyl C-atom of the adjacent A-methylvalinc (position 11) [86],... 

The mechanism of catalysis by these enzymes has been extensively investigated (for review see ref. 10). Essentially, the active site serine via its side chain hydroxyl group performs a nucleophilic attack on the carbonyl carbon of the scissile peptide bond thus forming a tetrahedral intermediate. A histidine residue in the active site serves as a general base accepting the proton from the serine residue. The acyl enzyme thus formed is broken down via a nucleophilic attack of a water molecule to complete the hydrolysis of the peptide bond. 

Tlie neurotransmitter acetylcholine is both a quaternary ammonium compound (see Box 6.7) and an ester. After interaction with its receptor, acetylcholine is normally degraded by hydrolysis in a reaction catalysed by the enzyme acetylcholinesterase. This enzyme contains a serine residue that acts as the nucleophile, hydrolysing the ester linkage in acetylcholine (see Box 13.4). This effectively acetylates the serine hydroxyl, and is an example of transesterification (see Section 7.9.1). For continuation of acetylcholine degradation, the original form of the enzyme must be regenerated by a further ester hydrolysis reaction. 

The mechanism of action of chymotrypsin can be rationalized as follows (Figure 13.5). The enzyme-substrate complex forms, with the substrate being positioned correctly through hydrogen bonding and interaction with the pocket as described above. The nucleophilicity of a serine residue is only modest, but here it is improved by... 

Note that penicillins and structurally related antibiotics are frequently deactivated by the action of bacterial -lactamase enzymes. These enzymes also contain a serine residue in the active site, and this is the nucleophile that attacks and cleaves the P-lactam ring (see Box 7.20). The P-lactam (amide) linkage is hydrolysed, and then the inactivated penicillin derivative is released from the enzyme by further hydrolysis of the ester linkage, restoring the functional enzyme. The mode of action of these enzymes thus closely resembles that of the serine proteases there is further discussion in Box 7.20. 

The site of action in the 3-lactam antibiotics is muramoylpentapeptide carboxypeptidase, an enzyme that is essential for cross-linking of bacterial cell walls. The antibiotic resembles the substrate of this enzyme (a peptide with the C-terminal sequence D-Ala-D-Ala) and is therefore reversibly bound in the active center. This brings the 3-lactam ring into proximity with an essential serine residue of the enzyme. Nucleophilic substitution then results in the formation of a stable covalent bond between the enzyme and the inhibitor, blocking the active center (see p. 96). In dividing bacteria, the loss of activity of the enzyme leads to the formation of unstable cell walls and eventually death. 

Many natural products are constrained by macrocyclic motifs, which are often essenhal for natural products to possess the desired biological properties. In the biosynthesis of macrocyclic NRPs and PKs, linear peptides or PKs are often mac-rocyclized by a TE domain located at the C-terminal of multi-modular synthases. For example, in the biosynthesis of the antibiotic tyrocidine A (Tyc A), a linear enzyme-bound decapephde, which is transferred from the last carrier protein (or thiolahon) domain of the Tyc A synthase, is cyclized by an intramolecular Sn2 reachon between the N-terminal amine nucleophile and the C-terminal ester, which is covalently linked to serine residual in the TE domain prior to macro-cyclization (Scheme 7.9) ([35] and references therein). 

In the acylation step a nucleophilic group on one of the amino-acid side chains at the active site behaves as the nucleophile. As we have seen in Section 25-9B, the nucleophile of carboxypeptidase is the free carboxyl group of glutamic acid 270. In several other enzymes (chymotrypsin, subtilisin, trypsin, elastase, thrombin, acetylcholinesterase), it is the hydroxyl group of a serine residue ... 

Serine Proteases Enzymes that Use a Serine Residue for Nucleophilic Catalysis Ribonuclease A An Example of Concerted Acid-Base Catalysis... 

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