Enzymatic active site of caspase-activated DNase (CAD) and its inhibition by inhibitor of CAD.
Caspase-activated DNase (CAD) is a deoxyribonuclease that causes DNA fragmentation during apoptosis. In proliferating cells, CAD is complexed with ICAD (inhibitor of CAD) and its DNase activity is suppressed. Here, we established a quantitative assay for CAD DNase that measures the number of 3' hydroxyl groups on the CAD-generated DNA fragments. Chemical modification of histidine residues and substrate protection experiments demonstrated the presence of reactive histidine residues within the active site of the enzyme. Analysis by site-directed mutagenesis suggested that at least four histidine residues in the C-terminal part of the molecule are essential for the catalytic activity of CAD DNase. ICAD did not protect CAD from the chemical modification of the histidine residues, indicating that it does not mask the active site of CAD. In contrast, ICAD blocked the ability of CAD to bind DNA, suggesting that ICAD causes steric or electrostatic hindrance in CAD for substrate DNA. This molecular mechanism for the inhibition of CAD DNase by ICAD is similar to that proposed for colicin endonuclease and its inhibitor, immunity protein.
Sakahira
H
H
Department of Genetics, Osaka University Medical School, Japan Science and Technology Corporation, Suita.
Takemura
Y
Y
Nagata
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000737:chemistry; D007093:Imidazoles / Q000494:pharmacology; D007313:Insecta; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D007093:Imidazoles; D011994:Recombinant Proteins; D006639:Histidine; C029899:imidazole; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; D020169:Caspases; D004047:Diethyl Pyrocarbonate