Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B
Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B
复制标题
DOI：
10.1021/bi027429i
复制
发表时间：
2003-05-20
期刊：
影响因子：
2.9
通讯作者：
Lipscomb, JD
中科院分区：
文献类型：
--
作者：
Brazeau, BJ;Lipscomb, JD
假设可溶性甲烷单加氧酶 (sMMO) 的调节成分 MMOB 通过形成针对甲烷和 O-2 调节的结构灵活性增加的尺寸特定通道或区域来控制底物进入羟化酶成分 (MMOH) 的活性位点。因此，四个 MMOB 残基（N107G/S109A/S110A/T111A，Quad 突变体）大小的减小被证明可以加速大于甲烷的底物与反应性 MMOH 中间体 Q 的反应 [Wallar, B. J., and Lipscomb, J. D. (2001) Biochemistry 40, 2220-2233]。在这里，通过构建涉及四突变体残基的单突变和双突变来检验该假设。结果表明，延伸至 MMOB 核心结构的残基突变改变了 MMOH 催化反应的许多方面，但不模仿 Quad 突变体的效果。相反，如在 Quad 突变体中观察到的那样，被认为形成 MMOH-MMOB 复合物界面一部分的 MMOB 残基增加了活性位点的可及性。特别是，突变体 T111A 模仿了 Quad 突变体的大部分效果；因此，建议使用Thr111来最直接地控制访问。出乎意料的是，Thr111 突变为较大的 Tyr 大大增加了较大底物（如乙烷、呋喃和硝基苯）与 Q 反应的速率常数，同时降低了与甲烷反应的速率常数。循环中的其他步骤显着减慢，硝基苯氧化的区域特异性发生改变，并且需要比野生型 MMOB 多 10 倍的 T111Y 才能最大限度地提高周转率。因此，T111Y 似乎在局部界面结构上发生了更广泛的变化，允许至少与乙烷一样大的碳氢化合物与 Q 类似地结合和反应。结果，首次显示甲烷、乙烷及其氘代类似物的键断裂速率与键强度相关，符合底物与 Q 反应期间发生 C-H 键断裂的机制。
The regulatory component MMOB of soluble methane monooxygenase (sMMO) has been hypothesized to control access of substrates into the active site of the hydroxylase component (MMOH) through formation of a size specific channel or region of increased structural flexibility tuned to methane and O-2. Accordingly, a decrease in the size of four MMOB residues (N107G/S109A/S110A/T111A, the Quad mutant) was shown to accelerate the reaction of substrates larger than methane with the reactive MMOH intermediate Q [Wallar, B. J., and Lipscomb, J. D. (2001) Biochemistry 40, 2220-2233]. Here, this hypothesis is tested by construction of single and double mutations involving the residues of the Quad mutant. It is shown that mutations of residues that extend into the core structure of MMOB alter many aspects of the MMOH catalyzed reaction but do not mimic the effects of the Quad mutant. In contrast, the MMOB residues that are thought to form part of the interface in the MMOH-MMOB complex increase active site accessibility as observed for the Quad mutant. In particular, the mutant T111A mimics most of the effects of the Quad mutant; thus, Thr111 is proposed to most directly control access. Unexpectedly, mutation of Thr111 to the larger Tyr greatly increases the rate constant for the reaction of larger substrates such as ethane, furan, and nitrobenzene with Q while decreasing the rate constant for the reaction with methane. Other steps in the cycle are dramatically slowed, the regiospecificity for nitrobenzene oxidation is altered, and 10-fold more T111Y than wild-type MMOB is required to maximize the rate of turnover. Thus, T111Y appears to make a more extensive change in local interface structure that allows hydrocarbons at least as large as ethane to bind and react with Q similarly. As a result, the bond cleavage rates for methane, ethane, and their deuterated analogues are shown for the first time to correlate with bond strength in accord with a mechanism in which C-H bond cleavage occurs during reaction of substrates with Q.