title: Caspase-9 holoenzyme is a specific and optimal procaspase-3 processing machine.
abstract: Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome, and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper-linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC but is much less active than C9Holo for the physiological substrate procaspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for procaspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for procaspase-3, which is important for procaspase-3 activation at the physiological concentration.
authors.affiliation: Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yin
authors.forename: Hyun Ho
authors.initials: HH
authors.lastname: Park
authors.forename: Jee Y
authors.initials: JY
authors.lastname: Chung
authors.forename: Su-Chang
authors.initials: SC
authors.lastname: Lin
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Lo
authors.forename: Li S
authors.initials: LS
authors.lastname: da Graca
authors.forename: Xuejun
authors.initials: X
authors.lastname: Jiang
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D006639:Histidine / Q000737:chemistry; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D020035:Holoenzymes; D006639:Histidine; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases