Department of Medicinal Chemistry and ‡Minnesota NMR Facility, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Biochemistry. 2013 May 21;52(20):3588-600. doi: 10.1021/bi301616c. Epub 2013 May 7.
Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate and acyl adenylate, with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy [Zimon, M., et al. (2012) Nat. Genet. 44, 1080-1083]. We have conducted the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double-displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate-limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKa values of 6.5 and 8, with the former pKa agreeing well with the nuclear magnetic resonance titration results for the pKa of the active site nucleophile His112. In comparison to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 10(6)-10(8)-fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His112, while being partially rate-limited by intermediate hydrolysis and product release associated with a conformational change. Given the high degree of sequence homology of Hint proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.
人类组氨酸三核苷酸结合蛋白 1（hHint1）是组氨酸三核苷酸蛋白超家族中普遍存在的古老分支的成员。hHint1 是一种同源二聚体蛋白，可高效催化模型底物磷酸酰胺和酰基腺苷酸的水解。最近，无催化活性的 hHint1 被鉴定为遗传性周围神经病的原因[Zimon，M.等人。（2012 年）自然遗传学 44,1080-1083]。我们对 hHint1 进行了首次详细的动力学机制研究，发现反应机制与双置换机制一致，其中活性位点亲核试剂 His112 首先被底物腺苷酰化，然后 AMP-酶中间产物水解。来自停流荧光测定的瞬态爆发阶段和线性阶段表明，酶腺苷酰化速度快于随后的中间水解和产物释放。溶剂粘度实验表明，化学转化和扩散敏感事件（产物释放或蛋白质构象变化）都限制了整体周转率。催化捕获实验和数据模拟表明，最终产物 AMP 的真实 koff 速率不太可能控制整体 kcat。因此，与产物释放相关的蛋白质构象变化可能是限速步骤。此外，还发现 Hint1 的腺苷酰化速率取决于两个 pKa 值为 6.5 和 8 的残基，前一个 pKa 值与活性位点亲核试剂 His112 的核磁共振滴定结果非常吻合。与无催化速率相比，hHint1 被证明可将酰基-AMP 和 AMP 磷酸酰胺水解分别增强 10（6）-10（8）倍。总的来说，我们的分析表明，hHint1 通过依赖于活性残基 His112 的快速腺苷酰化的机制，高效催化磷酸酰胺和酰基腺苷酸的水解，而中间水解和与构象变化相关的产物释放则部分限制了反应速率。鉴于 Hint 蛋白在所有生命领域的高度序列同源性，它们的动力学和催化机制很可能与 hHint1 阐明的机制相似。
