Catalytic dyad

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. 

Fig. 3.9. The Cys-thiolate/His-imidazolium catalytic dyad and the oxyanion hole of papain,... 

At least ten caspases are known and many observations have confirmed their role in apoptosis (see also Chapter 32). In caspases 1 and 2 the side chains of Cys 285 and His 237 form the catalytic dyad and peptide NH groups of Cys 285 and Gly 238 form the oxyanion hole.333... 

The most recently discovered group of proteases are the N-terminal threonine hydrolases of the multi-catalytic protease complex (MPC) of proteasomes. The enzymes are arranged in a regular array inside proteasomal compartments as shown in Box 7-A. The active site is a catalytic dyad formed from the amino group at the N terminus of the P subunits.345-3463... 

Catalytic dyad 620 Catalytic subunits 348 Catalytic triad 134, 611-614, 619, 635 of serine proteases 611—614 Cataracts 169 Catechol 838 Catecholamine(s) 553 Catechol O-methyltransferase (COMT) 591 Cathepsin(s) 619,621 G 610... 

The three-dimensional structure of NTE has not been experimentally determined, but a homology model of the patatin domain indicates that the active site serine (Ser ) is located on a nucleophilic elbow characteristic of serine hydrolases (Wijeyesakere et al., 2007). Moreover, the model indicates that the catalytic site of NTE consists of a novel Ser-Asp catalytic dyad, as in patatin and mammalian cytosolic phospholipase A2 (CPLA2), rather than the classical catalytic triad (Ser-Asp/Glu-His), as found in many serine hydrolases including AChE. Recently, mutations have been identified in NTE that are associated with motor neuron disease (Rainier et al., 2008). The mutations occur within the catalytic domain of NTE, but it is not yet known if they affect the catalytic function of the enzyme or alter some other property of the protein in order to produce disease. 

Figure 9 Rhomboids contain a conserved serine and histidine, which comprise a putative catalytic dyad of a serine protease. Rhomboid-1 cleaves within the transmembrane region of the Drosophila EGF-like growth factor Spitz.

Most recently, the first crystal structures of an I-CLiP have been reported by three different research groups, all on the E. coli Rhomboid GlpG (90-92). These structures show remarkable similarities and important differences that provide insight into how this class of membrane-embedded protease carries out hydrolysis in the lipid bilayer. The structures all reveal that the key serine and histidine implicated as the catalytic dyad indeed are coordinated with each other and lie at a depth within the membrane consistent with where Rhomboids cleave their... 

The enzyme glucosylasparaginase (aspartylgluco-saminidase) is one of a group of other enzymes that use N-terminal threonyl groups as catalytic dyads. 

The cleavage mechanism of the caspases is depicted schematically in Fig. 15.6. A typical protease mechanism is used, with a catalytic dyad for cleavage of the peptide bond. The nucleophilic thiol of Cys285 forms a covalent thioacyl bond to the substrate during the catalysis. The imidazole ring of His237 is also involved in the catalysis, facilitating hydrolysis of the amide bond by acid/base catalysis. 

Clan SC contains peptidases with the a/P hydrolase fold bearing the catalytic triad in the order Ser, Asp, His. This clan includes the families (characteristic member in parentheses) S9 (prolyl oligopeptidase), S10 (carboxypeptidase C), S15 (Xaa-Pro dipeptidyl -peptidase), S28 (lysosomal Pro-Xaa carboxypeptidase), S33 (prolyl amino-peptidase), and S37 (Streptomyces PS-10 peptidase). The characteristic catalytic dyad Ser, Lys of dan SE is represented by the motif Ser-Xaa-Xbb-Lys, and the fold consists of helices and an a + P sandwich. The families of this clan Sll (penicillin-binding protein 5), S12 (Streptomyces R61 D-Ala-D-Ala carboxypeptidase), S13 (penicillinbinding protein 4) are involved in the biosynthesis, turnover and lysis of bacterial cell walls. 

The catalytic residues in dan SF (catalytic dyad Ser, Lys or Ser, His) are more widely spaced in comparison with clan SE. The families of this clan include only endopepti-dases from bacteriophages, bacteria, archaea and eukaryotes with the members S24... 

Aspartic peptidases have so far been described for all endopeptidases. Unfortunately, the tertiary structure has only been elucidated for four families. Endopeptidases of the family A1 consist of two lobes, with the active site between them. One lobe has been derived from the other by gene duplication. In the active site each lobe, with very similar three-dimensional structures, bears one Asp residue of the catalytic dyad. It is interesting to note that the crystal structure of retropepsin from family A2 of clan AA showed a single lobe with one catalytic Asp residue with structural similarity to one lobe of the pepsin from family Al. Retropepsin is only active as a homodimer forming the catalytic site between the two monomeric molecules. There is evidence that the peptidases of families Al and A2 have evolved from a common ancestor. Unfortunately, a number of other families could not yet been assigned to any clan. 

GPIS is presumed to be the catalytic center of the enzyme since it shares sequence homology with a family of cysteine proteases, one member of which has transamidase activity in vitro. Mutagenesis of a Cys-His catalytic dyad in human GPIS inactivates GPIT. The functions of the remaining subunits are unknown. It seems likely that they recruit substrates, or regulate substrate access to the catalytic site (S. Vainauskas, 2004, 2006). 

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