Caspase-3/Sandbox: Difference between revisions
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==Structure & Function== | ==Structure & Function== | ||
===Structure=== | ===Structure=== | ||
[[Image:Rossman fold.png|350px|right]] | |||
Procaspase-3, unlike initiator procaspases, is a stable dimer with little enzymatic activity. Procaspase-3 contains a tri-aspartate ‘safety-catch’ (Asp179–Asp181) in the intersubunit linker to remain nonfunctional. During the process of activation, this linker undergoes a pH-dependent conformational change, exposing Asp175 for cleavage. This leads to the formation of a heterotetramer (Walters, 2011). The heterotetramer consists of two anti-parallel arranged heterodimers, each one formed by a 17 kDa (p17) and a 12 kDa (p12) subunit (http://www.uniprot.org/uniprot/P42574). | Procaspase-3, unlike initiator procaspases, is a stable dimer with little enzymatic activity. Procaspase-3 contains a tri-aspartate ‘safety-catch’ (Asp179–Asp181) in the intersubunit linker to remain nonfunctional. During the process of activation, this linker undergoes a pH-dependent conformational change, exposing Asp175 for cleavage. This leads to the formation of a heterotetramer (Walters, 2011). The heterotetramer consists of two anti-parallel arranged heterodimers, each one formed by a 17 kDa (p17) and a 12 kDa (p12) subunit (http://www.uniprot.org/uniprot/P42574). | ||
Caspase-3 chains are classified as alpha/beta, with one chain containing a 3-layer(aba) sandwich with Rossmann fold topology | Caspase-3 chains are classified as alpha/beta, with one chain containing a 3-layer(aba) sandwich with Rossmann fold topology and another chain containing a 2-layer sandwich with alpha-beta plaits. | ||
====Salt Bridge==== | ====Salt Bridge==== | ||
[[Image:Casp3Salt bridge.png| | [[Image:Casp3Salt bridge.png|400px|left]] | ||
Lys242 is located at the C-terminus of helix 5 and Glu246 is located at the base of loop L4 of caspase-3. It has been suggested that the salt bridge between Lys242-Glu246 (NH3+ of Lys with COO- of Glu) is not found in the zymogen form of caspase-3 but forms only upon caspase-3 maturation. Rather, these residues are involved in other interactions in procaspase-3. Lys242 maintains the native procaspase dimer, whereas glutamate is involved in stabilizing loop L3, the substrate binding loop. In procaspase-3, Glu246 participates in hydrogen bonding with Trp214. In the mature, active caspase-3, the stability of the active site of caspase-3 depends on the salt bridge between Lys242 and Glu246 to maintain the correct conformation of loop L4. In caspase-3, Glu246 neutralizes the positive charge of Lys242 by forming a salt bridge within the same hydrophobic cluster of residues between helices 4 and 5 that buries the aliphatic portion of Lys242. Mutations of either K242A or E246A resulted in the complete loss of procaspase-3 activity as measured by fluorescence emission following addition of substrate (Ac-DEVD-AFC) and excitation at 400nm. These mutants were active in the mature, fully cleaved caspase-3, although to a much lower extent than wild type mature caspase-3, perhaps due to altered accessibility of its active site. The measurements of kcat/Km for these mutants at the optimal pH (7.5) for caspase-3 activity are 14 to 60-fold lower than that of wild type, with an overall increase in Km and a decrease in kcat. This may be due to alterations caused by the mutation(s) in the loop bundle interaction between loops L2, L4 and L2’. Loss of the Lys242-Glu246 salt bridge may alter the position of helix 4, which consequently alter the position of loop L3, leading to altered hydrogen-bonding in the P4 site. This may explain the decrease in activity seen in the mutants. Furthermore, because Phe250 in loop L4 participates in hydrogen bonding to P4, mutations in loop L4 could result in disruptions of these interactions (Feeney, 2004; Walters, 2011). | Lys242 is located at the C-terminus of helix 5 and Glu246 is located at the base of loop L4 of caspase-3. It has been suggested that the salt bridge between Lys242-Glu246 (NH3+ of Lys with COO- of Glu) is not found in the zymogen form of caspase-3 but forms only upon caspase-3 maturation. Rather, these residues are involved in other interactions in procaspase-3. Lys242 maintains the native procaspase dimer, whereas glutamate is involved in stabilizing loop L3, the substrate binding loop. In procaspase-3, Glu246 participates in hydrogen bonding with Trp214. In the mature, active caspase-3, the stability of the active site of caspase-3 depends on the salt bridge between Lys242 and Glu246 to maintain the correct conformation of loop L4. In caspase-3, Glu246 neutralizes the positive charge of Lys242 by forming a salt bridge within the same hydrophobic cluster of residues between helices 4 and 5 that buries the aliphatic portion of Lys242. Mutations of either K242A or E246A resulted in the complete loss of procaspase-3 activity as measured by fluorescence emission following addition of substrate (Ac-DEVD-AFC) and excitation at 400nm. These mutants were active in the mature, fully cleaved caspase-3, although to a much lower extent than wild type mature caspase-3, perhaps due to altered accessibility of its active site. The measurements of kcat/Km for these mutants at the optimal pH (7.5) for caspase-3 activity are 14 to 60-fold lower than that of wild type, with an overall increase in Km and a decrease in kcat. This may be due to alterations caused by the mutation(s) in the loop bundle interaction between loops L2, L4 and L2’. Loss of the Lys242-Glu246 salt bridge may alter the position of helix 4, which consequently alter the position of loop L3, leading to altered hydrogen-bonding in the P4 site. This may explain the decrease in activity seen in the mutants. Furthermore, because Phe250 in loop L4 participates in hydrogen bonding to P4, mutations in loop L4 could result in disruptions of these interactions (Feeney, 2004; Walters, 2011). | ||