Figure 5. Caspase catalytic mechanism.
Close-up of the active-site region in acetyl-Asp-Val-Ala-Asp-methyl ketone-inhibited caspase-3 (PDB code 1CP3; [10]) shown in standard orientation, i.e. with the active-site residues facing the viewer, and substrates running from left to right. The stereo plots display (a) a ribbon representation of the caspase (large subunit, blue; small subunit, red), and (b) the GRASP electrostatic surface potential of the caspase (contoured between −25 and +25 kBT/e) with stick inhibitor. Important residues are labelled in both panels. Hydrogen bonds were calculated using HBPLUS (http://www.biochem.ucl.ac.uk/bsm/hbplus/home.html) and are indicated with orange dotted lines in (a). Note that the inhibitor binds in an extended conformation, with backbone atoms of P3 and P1 residues hydrogen-bonded to strictly (Arg-341) and highly (Ser-339) conserved caspase residues. The guanidinium groups of Arg-179 and Arg-341 engage in strong salt bridges with the carboxylate of the P1 aspartate, which is further hydrogen-bonded to the side-chain carboxyamide of Gln-283. The combination of extended, β-sheet-like hydrogen bonding to the enzyme and of substrate recognition based mainly on interactions with the S1 and S4 pockets places caspases in a mechanistic sense closer to serine proteases, in particular those of the subtilisin clan. (c) Proposed substrate-hydrolysis mechanistic scheme. During the acylation step (1), the carbonyl oxygen of the non-covalently bound P1 residue is anchored through hydrogen bonds to the nitrogen atoms of Gly-238 and Cys-285 (the oxyanion hole). This increases the polarization of the C–O bond, and therefore facilitates nucleophilic attack of the sulphur atom of Cys-285 on the highly electrophilic carbonyl carbon. The result is a covalent enzyme–substrate adduct, the high-energy tetrahedral intermediate (2), as visualized in crystal structures of methyl ketone-inhibited caspases (see a). The imidazole moiety of His-237 acts as a general acid at this stage of catalysis by protonating the α-amino group of the leaving peptide product, thus avoiding re-formation of the peptide bond. Deacylation of the acyl-enzyme complex occurs then in a similar manner: the deprotonated His-237 side chain abstracts a proton from a water molecule, the hydrolytic water (3), which is thus activated to attack the thioester bond. Deacylation proceeds through a second tetrahedral intermediate (4), formed upon nucleophilic attack of the hydroxy group on the carbonyl carbon. (A putative, neutral gem-diol intermediate found in a recent quantum mechanics/molecular mechanics simulation of the hydrolysis of the acyl-enzyme complex in caspase-3 [79] is shown by grey atoms in parentheses. These authors also predicted that the catalytic histidine is activated by the hydroxy group of Ser-178, but this residue is not conserved in other caspases.) Rupture of the Sγ–C bond regenerates the enzyme in a non-covalent complex with the N-terminal peptide product (5). By analogy with serine proteases, it is conceivable that movements of the 341- and/or 381- substrate-binding loops are coupled to the latter reaction, thus allowing disruption of the main-chain–main-chain hydrogen bonds with the P1/P3 residues, and of the P1 carbonyl oxygen atom with the oxyanion hole. In other words, thioester hydrolysis and product release may be synchronized to ensure a high efficiency of catalysis.