Cui Danica S, Broom Aron, Mcleod Matthew J, Meiering Elizabeth M, Holyoak Todd
Department of Biology and ‡Department of Chemistry, University of Waterloo , Waterloo, ON, Canada N2L 3G1.
Biochemistry. 2017 Apr 18;56(15):2106-2115. doi: 10.1021/acs.biochem.7b00178. Epub 2017 Apr 4.
Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ω-loop by 3.4 kcal mol. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predominant effect being observed in the direction of oxaloacetate synthesis. This is exemplified by a reduction in k for the H477R mutant by an order of magnitude in the direction of OAA synthesis, while in the direction of PEP synthesis, it decreased by a factor of only 2. The data are consistent with a mechanism for loop conformational exchange between open and closed states in which a balance between fixed anchoring of the N-terminus of the Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state.
可移动的Ω环在许多酶的功能中起着至关重要的作用。在此，我们利用晶体学、动力学和计算分析方法，研究了位于可移动Ω环元件之外的一个残基在胞质磷酸烯醇丙酮酸羧激酶H477R变体催化功能中的重要性。晶体学数据表明，Ω环向闭合构象的有效转变需要通过R461和E588之间的接触来稳定环的N端。相反，Ω环的C端通过环C端的H477与位于酶体上的E591之间的接触，与酶体发生不断变化的相互作用。平均力势计算表明，通过H477R取代改变Ω环C端的锚定，会导致Ω环闭合状态的稳定性降低3.4千卡/摩尔。该酶的动力学参数以不对称方式改变，主要影响在草酰乙酸合成方向上观察到。这体现在H477R突变体在OAA合成方向上的k值降低了一个数量级，而在PEP合成方向上仅降低了2倍。这些数据与Ω环在开放和闭合状态之间构象交换的机制一致，其中Ω环N端的固定锚定与灵活的、未连接的C端之间的平衡驱动了无序（开放）状态和有序（闭合）状态之间的转变。
