CO2 impairs peroxynitrite-mediated inhibition of human caspase-3.
Peroxynitrite (ONOO-) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide (.NO) with superoxide (O2.-). The peroxynitrite reactivity is modulated by carbon dioxide (CO2) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO2 on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the Sgamma atom of the Cys catalytic residue. In the absence of CO2, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO2 (=1.3x10(-3) M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the kcat value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering Km. Both in the absence and presence of CO2, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent the first evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO2, supporting the role of CO2 in fine tuning of cell processes (e.g., apoptosis).
Ascenzi
Paolo
P
Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146 Roma, Italy. ascenzi@uniroma3.it
Marino
Maria
M
Menegatti
Enea
E
D017209:Apoptosis; D002245:Carbon Dioxide / Q000378:metabolism*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002384:Catalysis; D049109:Cell Proliferation; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016877:Oxidants / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D016877:Oxidants; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D030421:Peroxynitrous Acid; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D004229:Dithiothreitol