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The Evolution and Mechanism of Enzyme Specificity and Stability of Caspase‐3
Author(s) -
Yao Liqi,
Clark Clay
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.779.50
Subject(s) - caspase , chemistry , salt bridge , dimer , effector , microbiology and biotechnology , caspase 1 , subfamily , proteases , biophysics , biochemistry , biology , enzyme , apoptosis , programmed cell death , mutant , organic chemistry , gene
The caspase family of proteases is an excellent model when analyzing protein evolution. Caspases are in a large family, and proteins evolved from a common ancestor. Caspases in the apoptotic initiator subfamily form a stable monomer, and their activities result from dimerization. Apoptotic effector caspases, however, can only form stable dimers. Urea folding/unfolding studies showed that human caspase‐3, an effector caspase, folds by a four‐state equilibrium process in which the native dimer (N2), unfolds through a non‐active dimeric intermediate (I2), a non‐native monomeric state (I) and an unfolded monomeric state (U). We compared the folding process of caspases from other species in order to examine whether the folding mechanism is conserved in other species. We show that zebrafish caspase‐3b also folds by the four‐state model. In addition, the human caspase‐3 dimer is stabilized by several salt bridges across the dimer interface, and dimerization is sensitive to pH due to the presence of two histidine residues in the salt bridges. In contrast, zebrafish caspase‐3a and ‐3b do not use histidines in the salt bridges, so the zebrafish dimers appear less sensitive to pH. Following evolution of the dimer, mutations in effector caspases resulted in changes in substrate specificity. The caspase‐6 subfamily shows preference to valine at the P4 position, while caspase‐3 shows preference to aspartate at the P4 position. However, recent research shows that zebrafish caspase‐3a has relaxed specificity and allows either valine and aspartate at the p4 position. In contrast, the closely related zebrafish caspase‐3b is similar to human caspase‐3 with ~100‐fold preference for D over V at P4. Our data shows that there are two ways to change enzyme specificity. The first one is through improving H‐bonds to the P4 aspartate by decreasing the H‐bond distance between carboxyl group in P4 and asparagine from active site loop 3. A second mechanism that results in selection of D over V is by including a polar cap near the S4 site. This mechanism is observed in caspase‐3b from zebrafish and appears to be a general strategy from the scaffold of the common ancestor. We show that mutations that break the salt bridge in human caspase‐3 results in relaxed specificity compared to wild type protein. In conclusion, protein conformational ensemble and enzyme specificity are two important ways to regulate caspase activity. Gaining the knowledge with regards to the mechanisms with which these proteins and their related families evolved may assist in uncovering how and why the various functions evolved in these proteins. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .