Serine esterases, mechanism

Acetylcholine is a relatively small molecule that is responsible for nerve-impulse transmission in animals. As soon as it has interacted with its receptor and triggered the nerve response, it must be degraded and released before any further interaction at the receptor is possible. Degradation is achieved by hydrolysis to acetate and choline by the action of the enzyme acetylcholinesterase, which is located in the synaptic cleft. Acetylcholinesterase is a serine esterase that has a mechanism similar to that of chymotrypsin (see Box 13.5). 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

The mechanism of action of anticholinesterases is to form a stable covalent complex with the Achase enzyme. Achase is one of several enzymes known as serine esterases. Other examples include the intestinal enzymes trypsin and chymotrypsin as well as the blood clotting agent thrombin. During the course of the catalysis the alcohol -OH of a serine side chain in the active site of the enzyme forms an ester complex, called the acyl-enzyme, with the substrate. So, acetylcholine will go through similar chemical reactions with Achase. 

Acetylcholinesterase (AChE) deesterifies the neurotransmitter acetylcholine (ACh). AChE belongs to a group of enzymes considered serine esterases and has a mechanism similar to that of chymotrypsin. AChE has an anionic binding site that attracts the positively charged quaternary ammonium group of ACh. The serine then attacks and cleaves the ester.910... 

Sperm penetrate the zona pellucida only after completion of the acrosome reaction. A similar process occurs in nonmammalian species, where sperm must penetrate the vitelline coat. In abalone this is accomplished by release of lysin, an acrosomal protein that disperses the vitelline coat by a noncatalytic mechanism (Lewis et al., 1982 Shaw et al., 1993). In contrast, the generally accepted model for mammalian sperm penetration of the zona pellucida is the acrosin hypothesis in which proteolysis of zona pellucida matrix glycoproteins by acrosin, the acrosomal serine esterase, plays a trailblazing role in the sperm penetration process (Yanag-... 

Serine carbohydrate esterases and transacylases. The commonest reaction mechanism is the standard serine esterase /protease mechanism, demonstrated paradigmally for chymotrypsin, involving an acyl-enzyme intermediate. The enzyme nucleophile is a serine hydroxyl, which is hydrogen bonded the imidazole of a histidine residue, whose other nitrogen is hydrogen bonded to a buried, but ionised, aspartate residue (Figure 6.28),... 

The previous discussion of amino acid catabolic disorders indicates that catabolic processes are just as important for the proper functioning of cells and organisms as are anabolic processes. This is no less true for molecules that act as neurotransmitters. To maintain precise information transfer, neurotransmitters are usually quickly degraded or removed from the synaptic cleft. An extreme example of enzyme inhibition illustrates the importance of neurotransmitter degradation. Recall that acetylcholine is the neurotransmitter that initiates muscle contraction. Shortly afterwards, the action of acetylcholine is terminated by the enzyme acetylcholinesterase. (Acetylcholine must be destroyed rapidly so that muscle can relax before the next contraction.) Acetylcholinesterase is a serine esterase that hydrolyzes acetylcholine to acetate and choline. Serine esterases have catalytic mechanisms similar to those of the serine proteases (Section 6.4). Both types of enzymes are irreversibly inhibited by DFP (diisopropylfluorophosphate). Exposure to DFP causes muscle paralysis because acetylcholinesterase is irreversibly inhibited. With each nerve impulse, more acetylcholine molecules enter the neuromuscular synaptic cleft. The accumulating acetylcholine molecules repetitively bind to acetylcholine receptors. The overstimulated muscle cells soon become paralyzed (nonfunctional). Affected individuals suffocate because of paralyzed respiratory muscles. 

Acetylcholinesterase is a serine esterase whose catalytic mechanism is similar to that of the serine proteases. As with chymotrypsin and trypsin, the active site of acetylcholinesterase has serine as part of a Ser-His-Asp catalytic triad. The mechanism -will involve covalent tetrahedral and acyl enzyme intermediates in which the substrate is bonded covalently to the active-site Ser. The reaction starts with nucleophilic attack on... 

The crystal structure analysis of PHB depolymerase from Penicillium funiculosum has been studied recently. " According to the structure described, the spatial arrangement of the catalytic residues in the enzyme indicates that the mechanism of the depol)onerase reaction may be similar to that of a lipase/ serine esterase. The overall catalytic mechanism is as shown in Figure 10.6. 

Both C3a and C5a have chemotactic activities as well (27,28). That is, they are capable of promoting migration of white blood cells to a site of inflammation. It is not clear whether the C5a and/or C3a fragments responsible for chemotaxis are chemically identical with the corresponding anaphylatoxin molecules. The precise mechanism by which chemotax-is is accomplished is only poorly understood at the present time, but it does appear that at least two serine esterases supplied by the polymoridio-nuclear leukocytes are required for this response to chemotactic factors. 

Considerahons of probable mechanisms of aging for mipafox-inhibited serine esterases suggest that mass spectrometry studies would support a deprotonation mechanism for NTE or NEST and hydrolytic P—N bond scission for AChE and BChE. As summarized in Figure 63.10, these expectahons were borne out for human recombinant NEST, which was used as a surrogate for NTE (Kropp et al., 2004) and BChE, respectively (Kropp and Richardson, 2007). However, mipafox-inhibited AChE gave the surprising result, confirmed by... 

Organophosphates phosphorylate the OH group of the catalytic serine at the active site of B-esterases (see Sect. 3.3). The rate of dephosphorylation of the enzyme is very slow, thus, the organophosphate acts as a mechanism-based inactivator. B-Esterases are classified as carboxylesterases (EC 3.1.1.1). 

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. 

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). 

Fig. 7.17 Proposed mechanism for non-specific esterase catalysis involving a serine residue.

All of these esterases appear to act by mechanisms closely related to those of proteases. Acetylcholinesterase contains an active site serine that reacts with organophosphorus compounds (Box 12-E) and is part of an Asp-His-Ser catalytic triad which lies in a deep "gorge" as well as an oxyanion hole.637 A surprise is the absence of an essential carboxylate group that might bind the positively charged trimethylammonium... 

Although the transition state analog approach is suitable for enzymes that bind their transition state noncovalendy, many natural enzymes achieve rate accelerations through covalent catalysis. For example, in the mechanism of most esterases and amidases, a functional group (e.g., a serine hydroxyl) of the protein covalently interacts with the substrate to form a protein bound intermediate. Furthermore, nature s most fundamental carbon-carbon bond-forming enzymes, class I aldolases, use... 

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