Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase
Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase
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DOI：
10.1021/bi052464l
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发表时间：
2006-04-04
期刊：
影响因子：
2.9
通讯作者：
Tanizawa, K
中科院分区：
文献类型：
--
作者：
Chiu, YC;Okajima, T;Tanizawa, K
铜胺氧化酶含有翻译后生成的醌辅因子，topa醌（TPQ），它介导电子从胺底物转移到分子氧。根据TPQ的氧化还原状态，将整个催化反应分为前还原半反应和后氧化半反应。在还原半反应中，底物胺与氧化TPQ的C5羰基反应，形成底物希夫碱（TPQ(ssb)），然后转化为产物希夫碱（TPQ(psb)）。在这个步骤中，假定一个不变的Asp残基具有较高的pK(a)，作为接受底物α质子的一般碱。当来自球形节杆菌的重组酶中假定的活性位点碱基Asp298突变为Ala时，催化效率下降到比野生型（WT）酶低约10(6)个数量级，与Asp298的必要性一致。对D298A突变体与2-苯基乙胺还原半反应过程中观察到的缓慢紫外/可见光谱变化进行全局分析，得出TPQ（ssb）的形成和衰变的表观速率常数(k（obs）分别为4.7和4.8 x 10(-4) s(-1))，这两个速率常数都明显小于快速扫描停流分析确定的WT酶（k（obs）分别为699和411 s-1）。因此，Asp298不仅在从TPQ（ssb）中提取a -质子中起着重要作用，而且在还原半反应的其他步骤中也起着重要作用。对D298A晶体浸泡1 h和1周的x射线衍射分析显示TPQ（ssb）和TPQ（psb）的结构分别为单晶显微分光光度法预先指定的结构。与α -质子提取的立体特异性一致，待提取TPQ（ssb）的pro-S α -质子几乎垂直于TPQ的希夫碱亚胺双键与醌环共轭形成的平面，使得共轭体系中sigma和pi电子轨道最大重叠。更有趣的是，即使在缺乏碱基的D298A突变体催化的反应中，底物的前s α质子也会被立体特异性释放。基于这些结果，我们提出，质子萃取的立体特异性主要取决于TPQ（ssb）的构象，而不是TPQ和催化碱的相对几何形状。
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 catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pK(a) is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A Mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s-1, respectively). Thus, Asp298 plays important roles not only in the a.-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.