Wang Mengmeng, Unruh Jay R, Johnson Carey K, Kuczera Krzysztof, Schowen Richard L, Borchardt Ronald T
Department of Chemistry, The University of Kansas, Lawrence, Kansas 66045, USA.
Biochemistry. 2006 Jun 27;45(25):7778-86. doi: 10.1021/bi0523106.
Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.
通过时间分辨荧光各向异性测量检测到了S-腺苷-L-高半胱氨酸（AdoHcy）水解酶的结构域运动。将天然酶中重定向运动的时间常数与通过定点突变改变关键残基的酶的时间常数进行了比较。在先前的正常模式研究[Wang等人（2005年）《一种酶的结构域运动和开放到闭合的构象转变：S-腺苷-L-高半胱氨酸水解酶的正常模式分析》，《生物化学》44卷，7228 - 7239页]中确定的酶的开放和闭合形式之间的铰链区中选择了突变M351P、H353A和P354A。在野生型无底物的AdoHcy水解酶（每个亚基中有NAD⁺辅因子）中，在10 - 20纳秒和80 - 90纳秒的时间尺度上检测到了重定向运动。较快的运动归因于结构域运动，较慢的运动归因于酶的翻滚。在酶复合物E（NADH/3'-酮基腺苷）和E（NAD⁺/3'-脱氧腺苷）中也检测到了结构域运动，但在复合物E（NADH/3'-酮基奈拉滨A）中未检测到。结果表明，AdoHcy水解酶以开放和闭合结构的平衡状态存在，对于无底物的酶E（NAD⁺）以及在底物结合后催化循环早期形成或在产物释放前后期形成的中间体E（NAD⁺/配体），平衡向更易移动的开放形式偏移。然而，强抑制剂奈拉滨A结合后会发生氧化，形成复合物E（NADH/3'-酮基奈拉滨）。对于这种类似于具有中心催化中间体的酶复合物的复合物，平衡向更刚性的闭合形式偏移。对于M351P和P354A突变体也观察到了类似的模式。相比之下，对于H353A蛋白，无论是否存在配体，或者辅因子处于氧化态还是还原态，都未检测到结构域运动，这表明该突变改变了酶的铰链弯曲动力学。
