Kinetic comparison of procaspase-3 and caspase-3.
Caspases, the key enzymes in apoptosis, are synthesized as proenzymes and converted into active form by proteolytic cleavage. The residues on active site reorganize during the activation process as shown in the comparative studies of crystallographic structures of procaspase-7 and its mature form. On the other hand, the proenzyme itself has some activity. Aiming to characterize the activation process, the comparative kinetic study for the pro- and mature caspase-3 was performed. In 1/K(M) versus pH study, a residue with pKa of 6.89+/-0.13 was detected only in caspase-3. While Vmax versus pH kinetic results were consistent with the existence of a residue with pKa of 6.21+/-0.06 in procaspase-3 mutant (D9A/D28A/D175A) but not in caspase-3. In the inactivation assays with diethylpyrocarbonate, a residue (pKa, 6.61+/-0.05) could be determined only for caspase-3 whereas with iodoacetamide a residue with pKa value (6.01+/-0.05) could be assigned only for procaspase-3. Considering that those residues could be protected by caspase-3-specific inhibitor from the inactivation, the modifiers are histidine- and cysteine-specific, respectively, and the involvement of these residues in the characteristic catalytic dyad of caspases, the results indicate that the pKa values of the catalytic histidine and cysteine residues are changed during the activation process.
Karki
Pratap
P
Research Center for Proteineous Materials, Department of Bio-Materials Engineering, Chosun University, Gwangju 501-759, Republic of Korea.
Lee
Jungsup
J
Shin
Song Yub
SY
Cho
Byungyun
B
Park
Il-Seon
IS
D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000737:chemistry; D007700:Kinetics; D009154:Mutation
D004792:Enzyme Precursors; D006639:Histidine; D053148:Caspase 3; D020169:Caspases; D003545:Cysteine; D004047:Diethyl Pyrocarbonate; D007460:Iodoacetamide