Control of catalytic cycle by a pair of analogous tRNA modification enzymes.
Control of catalytic cycle by a pair of analogous tRNA modification enzymes.
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DOI：
10.1016/j.jmb.2010.05.003
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发表时间：
2010-07-09
影响因子：
5.6
通讯作者：
Hou YM
中科院分区：
文献类型：
--
作者：
Christian T;Lahoud G;Liu C;Hou YM
使用不同活性位点结构进行相同反应的酶被称为类似酶。类似酶的分离表明存在多种酶结构途径可以催化相同的化学反应。关于类似酶的一个基本问题是，它们不同的活性位点结构是否会给化学反应带来相同或不同的动力学约束，特别是在酶周转的控制方面。在这里，我们用细菌TrmD及其真核和古细菌Trm5的类似酶来解决这个问题。TrmD和Trm5均以s -腺苷蛋氨酸（AdoMet）为甲基供体，催化甲基转移在tRNA反密码子附近的3'位置合成m1G37碱基，TrmD具有三叶结活性位点结构，而Trm5具有罗斯曼折叠结构。预稳态分析表明，TrmD的产物合成随时间呈线性增长，而Trm5的产物合成随时间呈快速增长，然后呈较慢的线性增长。Trm5的爆发动力学表明产物释放是催化循环的限速步骤，这与tRNA和AdoMet的产物具有较高的酶亲和力的观察结果一致。相反，缺乏爆发动力学的TrmD表明，它的周转是由产物合成所需的一个步骤控制的。虽然TrmD以同型二聚体的形式存在，但它对tRNA结合和产物合成表现出“半位点”反应性。TrmD和Trm5之间的动力学差异与两类氨基酰基-tRNA合成酶之间的动力学差异相似，它们使用不同的活性位点结构来催化tRNA氨基酰化。这种相似性表明，这些发现对于在解码过程中催化甲基和氨基酰基转移到tRNA的酶具有根本性的重要性。
Enzymes that use distinct active site structures to perform identical reactions are known as analogous enzymes. The isolation of analogous enzymes suggests the existence of multiple enzyme structural pathways that can catalyze the same chemical reaction. A fundamental question concerning analogous enzymes is whether their distinct active-site structures would confer the same or different kinetic constraints to the chemical reaction, particularly with respect to the control of enzyme turnover. Here we address this question with the analogous enzymes of bacterial TrmD and its eukaryotic and archaeal counterpart Trm5. While both TrmD and Trm5 catalyze methyl transfer to synthesize the m1G37 base at the 3' position adjacent to the tRNA anticodon, using S-adenosyl methionine (AdoMet) as the methyl donor, TrmD features a trefoil-knot active-site structure whereas Trm5 features the Rossmann fold. Pre-steady-state analysis revealed that product synthesis by TrmD proceeds linearly with time, whereas that by Trm5 exhibits a rapid burst followed by a slower and linear increase with time. The burst kinetics of Trm5 suggests that product release is the rate-limiting step of the catalytic cycle, consistent with the observation of higher enzyme affinities to the products of tRNA and AdoMet. In contrast, the lack of burst kinetics of TrmD suggests that its turnover is controlled by a step required for product synthesis. Although TrmD exists as a homodimer, it showed “half-of-the-sites” reactivity for tRNA binding and product synthesis. The kinetic differences between TrmD and Trm5 are parallel to those between the two classes of aminoacyl-tRNA synthetases, which use distinct active-site structures to catalyze tRNA aminoacylation. This parallel suggests that the findings have a fundamental importance for enzymes that catalyze both methyl and aminoacyl transfer to tRNA in the decoding process.
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影响因子：
16
作者：
Guth, Ethan C.;Francklyn, Christopher S.
通讯作者：
Francklyn, Christopher S.
影响因子：
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作者：
Hauenstein, Scott I.;Hou, Ya-Ming;Perona, John J.
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作者：
Flynn, J;Glickman, JF;Reich, NO
通讯作者：
Reich, NO
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作者：
Dupasquier, Marcel;Kim, Sangbumn;Hou, Ya-Ming
通讯作者：
Hou, Ya-Ming
影响因子：
2.9
作者：
Christian, Thomas;Evilia, Caryn;Hou, Ya-Ming
通讯作者：
Hou, Ya-Ming