Biochemical study on activation mechanism of caspases-3, -4, -9 and Bid cleavage by caspase-2

Abstract

Caspases, an evolutionarily conserved family of cysteine proteases, are the key mediators of apoptotic cell death program. The knowledge on the activation mechanism of these proteases provides the basis to understand the regulation of whole apoptotic machinery. In this study, the biochemical mechanism of activation of caspases-3, -4 and -9 was elucidated in terms of representative members of effector, inflammatory and initiator groups of caspases, respectively. During the comparative kinetic study between pro- and mature form of caspase-3, the enzymes showed identical KM values but procaspase-3 had ~ 200 times lower kcat than its mature form. Both of the enzymes also exhibited similar optimum pH values. However, in 1/KM vs. pH study, a residue with pKa of 6.89±0.13 was detected only for caspase-3 and Vmax vs. pH kinetic results were consistent with the existence of a residue with pKa of 6.21±0.06 in procaspase-3 mutant (Asp9, Asp28 and Asp175/Ala). In diethylpyrocarbonate inactivation assays, a residue with pKa of 6.61±0.05 was determined for caspase-3, whereas a residue with pKa of 6.01±0.05 was assigned for procaspae-3 with iodoacetamide. These results indicate that the pKa values of catalytic cysteine and histidine residues are changed during the activation process of caspase-3. With citrate, a well-known kosmotrope to enhance the monomer-dimer transition, caspase-4 was activated ~40 times that was comparable with that of caspase-9 (~75 fold increments). The activation reaction was mainly bimolecular ( 1.670.04) for monomeric caspase-4. The interdomain cleavage was responsible to activate caspase-4 more than 100-fold, again comparable with that of effector caspases. These results suggest that caspase-4 shows a novel activation mechanism of the synergism between dimerization and proteolysis. In this study, the constitutively dimeric forms of procaspase-9 were constructed by connecting the termini of two caspase-9 molecules. The dimers could be overexpressed and purified from Escherichia coli. The recombinant dimers showed two-fold increment in specific activity over their respective monomer that was lower than expected if dimerization was the only mechanism to activate caspase-9. The failure to gain the substantial catalytic advancements by the dimers suggests a different scenario of dimerization between the constructed dimers and Apaf-1 activated caspase-9. On analyzing the influence of biochemical parameters on caspase-2 activity, Bid, a substrate of caspase-8, was cleaved about one-fourth less efficiently by caspase-2 and it also cleaved procaspase-7 much weakly but not procaspase-3. In contrast to the optimal pH determined for its synthetic substrate, caspase-2 cleaved Bid and procaspase-7 much more efficiently at lower pH, while caspase-8 was more active at pH ≥7.0. Unlike the tolerance to 0-150 mM NaCl in hydrolyzing synthetic substrates, both caspases were sensitive towards higher concentration of salt in cleaving proteins. These observations suggest that the activity of caspase-2 for its physiological substrates is largely influenced by pH which might be a possible reason behind the inconsistency to observe the cleavage of its established in vitro substrates including Bid during caspase-2-induced apoptosis.