Catalytic serine

As an alternative to peptidic inhibitors, which display electrostatic interactions with the active site, covalent inhibitors have also been described recently. Such peptides bear a functional group that can react reversibly with the catalytic serine of the protease. These include aldehydes, a-ketoacid derivates, lactams and boronates. 

Thus, neither halogen substitution nor ring strain induces enzymatic hydrolysis. Molecules 21 and 22 may be bound in such a way that the (3-lactam carbonyl lies too far away from the catalytic serine hydroxyl group.72... 

The closest organic specie to the inorganic boric acid are the boronic acids generally described as R-B(OH)2. Boronic acids have been shown to act as inhibitors of the subtilisins. X-ray crystallographic studies of phenylboronic acid and phenyl-ethyl-boronic acid adducts with Subtilisin Novo have shown that they contain a covalent bond between the oxygen atom of the catalytic serine of the enzyme and the inhibitor boron atom (Matthews et al, 1975 and Lindquist Terry, 1974). The boron atom is co-ordinated tetrahedrally in the enzyme inhibitor complex. It is likely that boric acid itself interacts with the active site of the subtilisins in the same manner. 

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). 

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). 

To rationalize the stereospecificity of PLE toward a large variety of monocarboxylic and dicarboxylic esters, Tamm and co-workers have proposed the general formula displayed in Fig. 7.5 [5 5] [67]. Here, no representation of the active site is implied, but the model does rationalize numerous data and allows some qualitative predictions. A qualitative topographical model of the active site of PLE has been proposed by Jones and co-workers [68] [69], As shown in Fig. 7.6, substrate binding is defined by a carboxylate group that interacts with the catalytic serine residue, and by one or two hydrophobic groups that bind to sites 1 and/or 2. 

Fig. 18. The active site region of the electron density difference map between N-carbobenzoxy-L-alanine-elastase at —SS C and native elastase at the same temperature. The resolution is 3.5 A. The bilobed feature is consistent with the binding of the alanyi portion of the substrate to the oxygen of the catalytic serine, with weak interaction of the carbobenzoxy group to the surface of the enzyme.

Serine proteases are characterized by a catalytic triad of Ser, His, and Asp. They work in pairs of Ser-His and His-Asp. Replacement of Asp by a second His revealed the auxiliary nature of aspartate, probably in orienting the histidine with respect to the catalytic serine. All of the catalytic steps are performed by the dyad serine-histidine. 

Class D /3-lactamases show specificity toward oxacillins and are therefore named oxacillinases. In this class, the catalytic serine is activated by an N-carboxylated lysine <2001PNA14280, 2003PSC82>. 

The greatly increased nucleophilicity of the catalytic serine distinguishes it from all other serine residues and makes it an ideal candidate for modification via activity-based probes [58]. Of the electrophilic probe types to profile serine hydrolases, the fluorophosphonate (FP)-based probes are the most extensively used and were first introduced by Cravatt and coworkers [38, 39]. FPs have been well-known inhibitors of serine hydrolases for over 80 years and were first applied as chemical weapons as potent acetylcholine esterase inhibitors. As FPs do not resemble a peptide or ester substrate, they are nonselective towards a particular serine hydrolase, thus allowing the entire family to be profiled. FPs also show minimal cross-reactivity with other classes of hydrolases such as cysteine-, metallo-, and aspartylhydrolases [59]. Furthermore, FP-based probes react only with the active serine hydrolase, and not the inactive zymogen, allowing these probes to interact only with functional species within the proteome [59]. Extensive use of this probe family has demonstrated their remarkable selectivity for serine hydrolases and resulted in the identification of over 100 distinct serine hydrolases... 

The formation of a tetrahedral hemiacetal adduct was analyzed for the interaction between the inhibitor aldehyde and the catalytic serine residue (18)35>. The overall dissociation constants for an enzyme and an interacting transition state analog may be given by ... 

Trimethylammonium trifluoroacetophenone (19) was found to be a highly effective inhibitor of acetylcholinesterase 37 f The ketone activated by an electron-withdrawing trifluoroacetyl group will enhance the tendency to add a nucleophile (the hydroxyl group of the catalytic serine residue of acetylcholinesterase) to form a tetrahedral adduct as an aldehyde inhibitor. 

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

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. 

Clan SC peptidases are a/p hydrolase-fold enzymes that consist of parallel P-strands surrounded by a-helices. The a/p hydrolase-fold provides a versatile catalytic platform that, in addition to achieving proteolytic activity, can either act as an esterase, lipase, dehalogenase, haloperoxidase, lyase, or epoxide hydrolase (18). Six phylogenetically distinct families of clan SC are known, and oifly four of them have known structure. Catalytic amenability of the a/p hydrolase-fold may underlie why clan SC peptidases are the second largest family of serine peptidases in the human genome. Other mechanistic classes need not use the catalytic serine and instead use cysteine or glutamic acid (19). Clan SC peptidases present an identical geometry to the catalytic triad observed in clans PA and SB, yet this constellation is ordered differently in the polypeptide sequence. Substrate selectivity develops from the a-helices that surround the central P-sheet core. Within clan SC, carboxypeptidases from family SIO are unique for their ability to maintain... 

Fig. 5. A representation of the relative dispositions of the side chains of the catalytic aspartates and histidines in (A) RmL, (B) trypsin, (C) subtilisin, and (D) hPL. (A-C) the view is parallel to the plane of the imidazole, looking from where the catalytic serine would be, and along the H bond of His(N81)—Asp(OS2). The histidine nitrogens and the carboxyl oxygens of Asp are identified in A and are the same in B and C. (D) The view is rotated 90° with respect to the preceding view so that the interaction of Asp and His in hPL can be better visualized.

Molecular modeling studies were performed using the crystal structure of human elastase (pdb code IHNE), Sybyl molecular modeling software and a Tripos forcefield. An assumption was made that the silicon diol forms a covalent bond with the catalytic serine residue, Serl95. 

Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G. and Cambillau, C. (1992) Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature 356, 615-618... 

Figure 19. Structure of the active model of hPL with some selected side chains in yellow. The top three aromatic side chains have altered conformations, the segment containing Phe77 at the right has been shifted by 1.5 A away from the catalytic serine. The model of the substrate transition state is displayed in pink, the resulting product ester in red. The conformation of the fatty acid chains has been arbitrarily chosen to be extended.

Figure 23. Ca-display of the crystal structure of AChE in yellow with the central sheet in blue, the catalytic triad in green, and the central helix starting at the catalytic serine in pink.

Several structures of small molecule complexes with acetylcholinesterase have been solved. They reveal a binding site next to the catalytic serine preferrentially occupied by a positively charged moiety next to a hydrophobic portion. The positively charged functional groups almost superimpose in front of a trj tophan residue at the bottom of the gorge [25-27]. 

Figure 7. Schematic representation of the design principles. The rotation around C3-C4 is hindered, access of water is blocked, the catalytic serine remains acylated, and the enzyme is inactivated.

---