Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha.
Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha.
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DOI：
10.1021/bi901933d
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发表时间：
2010-03-23
期刊：
影响因子：
2.9
通讯作者：
Wilmot, Carrie M.
中科院分区：
文献类型：
--
作者：
Chang, Cindy M.;Klema, Valerie J.;Johnson, Bryan J.;Mure, Minae;Klinman, Judith P.;Wilmot, Carrie M.
在来自汉逊酵母的一对铜胺氧化酶 (CAO)（HPAO-1 和 HPAO-2）中研究了酶底物特异性的结构基础。提供了第二种铜胺氧化酶 (HPAO-2) 的 X 射线晶体结构（分辨率为 2.0 Å）和稳态动力学数据，以便与 HPAO-1 进行比较。尽管有 34% 的序列同一性和与催化有关的可叠加的活性位点残基，但这些酶的底物进入通道差异很大。先前研究的 CAO，HPAO-1，具有狭窄的底物通道。相比之下，HPAO-2 具有宽漏斗形底物通道，其中还包含一个侧室。此外，HPAO-2 和 HPAO-1 通道内存在许多氨基酸变化，可能会在空间上影响底物形成共价席夫碱催化中间体和引发化学反应的能力。这些差异可以部分解释以 kcat/Km 值差异为特征的底物特异性的巨大差异：在 HPAO-1 中，甲胺的 kcat/Km 比苄胺大 330 倍，而在 HPAO-2 中，苄胺是更好的底物 750 倍。在 HPAO-2 中，相对于 Dkcat/Km（苄胺）而言，膨胀的 Dkcat/Km（甲胺）表明质子提取受到的阻碍大于底物释放的阻碍。在 HPAO-1 中，Dkcat/Km(S) 随着底物速度慢而变化很小，表明控制底物结合和后续催化的能垒也有类似的增加。在这两种情况下，第二底物 O2 的 kcat/Km 都没有显着改变。这些结果强化了 CAO 活性位点的模块化性质，并表明多种因素有助于底物特异性和催化效率。在 HPAO-1（具有较小底物结合袋的酶）中，初始底物结合和质子损失均受到底物尺寸增加的影响，而在 HPAO-2（具有较大底物结合袋的酶）中，当底物中的苯基取代基减小到甲基大小时，质子损失率会受到不同的影响。
The structural underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 Å resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to HPAO-1. Despite 34 % sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast HPAO-2 has a wide funnel-shaped substrate channel, which also contains a side-chamber. In addition, there are a number of amino acid changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by kcat/Km value differences: in HPAO-1, the kcat/Km for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2 it is benzylamine that is the better substrate by 750-fold. In HPAO-2 an inflated Dkcat/Km(methylamine) in relation to Dkcat/Km(benzylamine) indicates that proton abstraction has been impeded more than substrate release. In HPAO-1, Dkcat/Km(S) changes little with the slow substrate, and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is kcat/Km for the second substrate, O2, significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with the smaller substrate binding pocket, both initial substrate binding and proton loss are affected by an increase in substrate size, while in HPAO-2, the enzyme with the larger substrate binding pocket, the rate of proton loss is differentially affected when a phenyl substituent in substrate is reduced to the size of a methyl group.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
8
作者：
Airenne, TT;Nymalm, Y;Salminen, TA
通讯作者：
Salminen, TA
影响因子：
2.6
作者：
DEHOOP, MJ;VALKEMA, R;AB, G
通讯作者：
AB, G
影响因子：
7.3
作者：
Marti, L;Abella, A;Zorzano, A
通讯作者：
Zorzano, A
影响因子：
5.7
作者：
Kumar, V;Dooley, DM;Zubak, VM
通讯作者：
Zubak, VM