Serine peptidases, mechanism

The mechanism by which serine peptidases, particularly serine endopep-tidases (EC 3.4.21), hydrolyze peptide bonds in peptides and proteins has been extensively investigated by X-ray crystallography, site-directed mutagenesis, detection of intermediates, chemical modification, H-NMR spectroscopy, and neutron diffraction [2-14], These studies revealed that all serine peptidases possess a catalytic triad, composed of a serine, a histidine, and an aspartate residue, and a so-called oxyanion hole formed by backbone NH groups. 

It is interesting to note that serine peptidases can, under special conditions in vitro, catalyze the reverse reaction, namely the formation of a peptide bond (Fig. 3.4). The overall mechanism of peptide-bond synthesis by peptidases is represented by the reverse sequence f-a in Fig. 3.3. The nucleophilic amino group of an amino acid residue competes with H20 and reacts with the acyl-enzyme intermediate to form a new peptide bond (Steps d-c in Fig. 3.3). This mechanism is not relevant to the in vivo biosynthesis of proteins but has proved useful for preparative peptide synthesis in vitro [17]. An interesting application of the peptidase-catalyzed peptide synthesis is the enzymatic conversion of porcine insulin to human insulin [18][19]. 

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). 

The mechanism of hydrolysis of cysteine peptidases, in particular cysteine endopeptidases (EC 3.4.22), shows similarities and differences with that of serine peptidases [2] [3a] [55 - 59]. Cysteine peptidases also form a covalent, ac-ylated intermediate, but here the attacking nucleophile is the SH group of a cysteine residue, or, rather, the deprotonated thiolate group. Like in serine hydrolases, the imidazole ring of a histidine residue activates the nucleophile, but there is a major difference, since here proton abstraction does not appear to be concerted with nucleophilic substitution but with formation of the stable thiolate-imidazolium ion pair. Presumably as a result of this specific activation of the nucleophile, a H-bond acceptor group like Glu or Asp as found in serine hydrolases is seldom present to complete a catalytic triad. For this reason, cysteine endopeptidases are considered to possess a catalytic dyad (i.e., Cys-S plus H-His+). The active site also contains an oxyanion hole where the terminal NH2 group of a glutamine residue plays a major role. 

Peptidases or proteases are enzymes that hydrolyse peptide bonds [9]. Proteolytic enzymes can be classified in five classes on basis of their catalytic mechanism aspartic, metallo-, cysteine, threonine and serine peptidases, whereby the latter three follow the same basic mechanism (Scheme 7.3) [10], Another classification of peptidases on the basis of statistically significant similarities in amino acid sequences was presented by Rawlings et al. (MEROPS database) [11], Serine proteases (SP) alone cover approximately one-third of all known proteases, and can accelerate the peptide hydrolysis very efficiently 10 fold) [6,11,12], SPs also hydro-... 

Several drugs in current medical use are mechanism-based enzyme inactivators. Eor example, the antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidoglycan chains during synthesis of bacterial cell walls (Eigure 14.17). Once cell wall synthesis is blocked, the bacterial cells are very susceptible to rupture by osmotic lysis, and bacterial growth is halted. 

The functionalized phenaceturates 16 (Fig. 11.10) are substrates of class A and C [3-lactamases, especially the class C enzymes, as observed with the parent unfunctionalized phenaceturates 15. They are also modest inhibitors of these enzymes and the serine DD-peptidase of Streptomyces R61. The inhibition of class C [3-lactamases is turnover dependent, as expected for a mechanism-based inhibitor. Inhibition is not very dependent on the nature of the leaving group, suggesting that the QM is generated in solution after the product phenol has been released from the active site. It therefore... 

Polgar, L. Catalytic mechanisms of serine and threonine peptidases. In Handbook of Proteolytic Enzymes, 2nd edition Barrett, A. J. Rawlings, N. D. Woessner, J. F., Eds. Elsevier Academic Press Amsterdam, Boston, Heidelberg, London, New York, Oxford, Paris, San Diego, San Francisco, Singapore, Sydney, Tokyo 2004, pp 1441-1448. 

The peptidases were separated into catalytic types according to the chemical nature of the group responsible for catalysis. The major catalytic types are, thus, Serine (and the related Threonine), Cysteine, Aspartic, Metallo, and As-Yet-Unclassified. An in-depth presentation of catalytic sites and mechanisms, based on this classification, is the subject of Chapt. 3. 

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. 

Fig. 3.3. Major steps in the hydrolase-catalyzed hydrolysis of peptide bonds, taking chymo-trypsin, a serine hydrolase, as the example. Asp102, His57, and Ser195 represent the catalytic triad the NH groups of Ser195 and Gly193 form the oxyanion hole . Steps a-c acylation Steps d-f deacylation. A possible mechanism for peptide bond synthesis by peptidases is represented by the reverse sequence Steps f-a.

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. 

This text is a good source of information on the chemical mechanisms underlying the different modes of peptidase catalysis. Three important enzymes are covered subtilisin, a serine endopepti-dase papain, a cysteine endopeptidase and chymosin, an aspartic endopeptidase. 

The results of a computational study revealed that the sedolisins, a family of ser-inecarboxyl peptidases, may evoke different catalytic machineries than do classical serine proteases in achieving transition-state stabilization. The family is characterized by a unique catalytic triad, Ser-Glu-Asp, that operates primarily through a general acid-base mechanism.76... 

The structures of hepatitis A viral 3C proteinases complexed with tetrapeptidyl-based methyl ketone inhibitors were shown to have an episulfide cation embedded in them. The authors concluded that the mechanism of inactivation of 3G peptidases by methyl ketone inhibitors is different than those operating in serine proteinases or in papain-like cysteine peptidases <2006MI673>. 

Yamamoto et al. [4] showed that 0.01% aprotinin (a serine protease inhibitor) reduced the metabolism of insulin and proinsuHn in homogenates of albino rabbit buccal mucosa, which otherwise would have occurred at 70-80% within 2.5 hours. Moreover, Lehr et al. suggest that polycar-bophil, a bioadhesive polymer, may protect some peptides from proteolysis, though the mechanism of this is unknown [5]. Others [6] have developed a series of pro-dmgs for peptides, with the aim of overcoming the metabohc barrier imposed by different peptidases. Stable prodrugs proved to be N-hydroxymethylated derivatives of the assessed dipeptides Gly-L-Leu and Gly-L-Ala [6]. 

The extensive use of penicillins and other P-lactam antibiotics in medicine over the last forty years has given rise to an increasing number of resistant bacteria. This bacterial resistance is mainly due to the production of P-lactamases [8], which efficiently catalyse the hydrolysis of the P-lactam ring. Like D,D-peptidases, most of the P-lactamases are serine proteases operating via the same mechanism as the transpeptidases. However, for P-lactamase activity k3 must be much larger than k2/K (Scheme 2). Recently, a structural homology between the two classes of bacterial enzymes has been established, suggesting that P-lactamases are evolutionary descendants of the D,D-peptidases [9,10]. 

Proteins can be modified by a group of peptide hydrolyses (peptidases) commonly called proteases (or proteinoses). Based on their ability to hydrolyze specific proteins, proteases are classified as collagenase, keratinase, elastase, etc. On the basis of the pH range over which they are active, they are classified as either acidic, neutral, or alkaline. However, according to their mechanism of action, the Enzyme Commission classifies proteases into the four distinct classes of serine, cysteine, aspartyl, and metalloproteases. Serine proteases, for example, always contain serine residue at their catalytic center, which is essential for the action of proteolysis. 

Peptidases including keratinases are hydrolases able to hydrolyze peptide bonds in proteins and peptides. They are classified using three different approaches (1) the chemical mechanism of catalysis (based on the catalytic amino acid or metal ion at then-active site, represented by serine, cysteine, threonine, aspartic, asparagine, glutamic and metallocatalytic type), (2) the catalytic reaction (this type of classification depends on the selectivity for the bonds that the peptidases will hydrolyze), and (3) the molecular structure and homology. In this latter approach, amino acid... 

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