Structure and Enzymatic Activity of Caspases

Caspases are expressed as inactive proenzymes (zymogens) and have to be activated by cleavage at internal conserved Asp residues. The proenzymes contain a N-terminal prodomain of various size, which in the case of Caspase-8 and Caspase-10 contains the DEDs (Death Effector Domains) that mediate the interaction with FADD/MORT1. This N-terminal prodomain is separated from the central large caspase-subunit (about 20 kDa: so called p20 subunit) by one or two Asp cleavage site(s). The large caspase-subunit itself is separated from the C-terminal small subunit (about 10 kDa: so called p10 subunit) by one Asp cleavage site or a linker peptide (two Asp cleavage sites). Caspases can activate themselves by autoproteolysis, or can be processed by other active caspases. At least in the case of Caspase-1, Caspase-8 and probably Caspase-10, the large prodomains are involved in the activation process of the caspases, while the function of the other prodomains is not yet known. The proteolytic cleavage leads to the formation of the active caspases which consist of the p10 and p20 subunits. The X-ray structure of Caspase-1 and Caspase-3 in complex with specific tetrapeptide inhibitors was elucidated. Their three-dimensional structures are comparable: the active proteins are tetramers of two p20 subunits surrounding two adjacent p10 subunits. Both, the p20 and p10 subunits are essential for catalytic activity. The active site pentapeptide QAC(R,G,Q)G is in the p20 subunit and forms the primary recognition pocket (S1) for the Asp residue, but several residues in the p10 and p20 subunits contribute to the specific binding of the substrate (tetrapeptide inhibitor) and form the recognition pockets S2-S4. The central cystein residue in the active site pentapeptide and a histidine residue form a catalytic diad, i.e. they directly contribute to the formation of the tetrahedral intermediate state in the hydrolytical cleaving mechanism of a substrate peptide by the caspases. While the active site pentapeptide (S1) is common to all caspases (they all are specific to Asp residue in the P1 position), the caspases differ in their recognition sites S2-S4 what explains their selectivity towards different cleavage motifs (= different residues in P2-P4 positions) and by this their substrate-specificity.