Kinetic and Structural Studies on the Catalytic Role of the Aspartic Acid Residue Conserved in Copper Amine Oxidase
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- 作者：Yen-Chen Chiu ; Toshihide Okajima ; Takeshi Murakawa ; Mayumi Uchida ; Masayasu Taki ; Shun Hirota ; Misa Kim ; Hiroshi Yamaguchi ; Yoshiaki Kawano ; Nobuo Kamiya ; $ ; Shun'ichi Kuroda ; Hideyuki Hayas
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：April 4, 2006
- 年：2006
- 卷：45
- 期：13
- 页码：4105 - 4120
- 全文大小：767K
- 年卷期：v.45,no.13(April 4, 2006)
- ISSN：1520-4995
文摘
Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone(TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalyticreaction is divided into the former reductive and the latter oxidative half-reactions based on the redoxstate of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of theoxidized TPQ, forming the substrate Schiff base (TPQssb), which is then converted to the product Schiffbase (TPQpsb). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as ageneral base accepting the 
proton of the substrate. When Asp298, the putative active-site base in therecombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency droppedto a level of about 106 orders of magnitude smaller than the wild-type (WT) enzyme, consistent with theessentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductivehalf-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for theformation and decay of TPQssb (kobs = 4.7 and 4.8 × 10-4 s-1, respectively), both of which are markedlysmaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (kobs = 699 and411 s-1, respectively). Thus, Asp298 plays important roles not only in the -proton abstraction fromTPQssb but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystalssoaked with the substrate for 1 h and 1 week revealed the structures of TPQssb and TPQpsb, respectively,as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of -protonabstraction, the pro-S -proton of TPQssb to be abstracted is positioned nearly perpendicularly to theplane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that theorbitals of 
and 
electrons maximally overlap in the conjugate system. More intriguingly, the pro-S proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lackingD298A mutant. On the basis of these results, we propose that the stereospecificity of -proton abstractionis primarily determined by the conformation of TPQssb, rather than the relative geometry of TPQ and thecatalytic base.
proton of the substrate. When Asp298, the putative active-site base in therecombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency droppedto a level of about 106 orders of magnitude smaller than the wild-type (WT) enzyme, consistent with theessentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductivehalf-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for theformation and decay of TPQssb (kobs = 4.7 and 4.8 × 10-4 s-1, respectively), both of which are markedlysmaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (kobs = 699 and411 s-1, respectively). Thus, Asp298 plays important roles not only in the -proton abstraction fromTPQssb but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystalssoaked with the substrate for 1 h and 1 week revealed the structures of TPQssb and TPQpsb, respectively,as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of -protonabstraction, the pro-S -proton of TPQssb to be abstracted is positioned nearly perpendicularly to theplane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that theorbitals of and electrons maximally overlap in the conjugate system. More intriguingly, the pro-S proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lackingD298A mutant. On the basis of these results, we propose that the stereospecificity of -proton abstractionis primarily determined by the conformation of TPQssb, rather than the relative geometry of TPQ and thecatalytic base.