Mutability of an HNH nuclease imidazole general base and exchange of a deprotonation mechanism
Mutability of an HNH nuclease imidazole general base and exchange of a deprotonation mechanism
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DOI：
10.1021/bi700418d
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发表时间：
2007-06-19
期刊：
影响因子：
2.9
通讯作者：
Stoddard, Barry L.
中科院分区：
文献类型：
--
作者：
Eastberg, Jennifer H.;Eklund, Jennifer;Stoddard, Barry L.
在自然界中已经独立地出现了几种催化磷酸二酯键水解的独特蛋白质折叠，包括PD（D/E）XK超家族（以II型限制性内切酶和许多重组和修复酶为代表）和HNH超家族（在同样广泛的酶中发现，包括细菌大肠杆菌素和归巢内切酶）。尽管PD（D/E）XK超家族中催化残基的身份和位置是高度可变的，但HNH核酸酶的活性位点更加保守。在这项研究中，HNH核酸酶的能力，以容忍其最保守的催化残基（其组氨酸一般基地）的突变，最活跃的酶变体的机制，其特征在于。将该残基转化为几种改变的化学物质，谷氨酰胺，赖氨酸或谷氨酸盐，导致可测量的活性。组氨酸到谷氨酰胺突变体显示出最高的残留活性和类似于野生型酶的pH曲线。该活性取决于相邻咪唑环的存在，该咪唑环已作为反应的较低效率的通用碱而被取代。这一结果意味着替代HNH衍生的催化位点的突变途径确实存在，但在性质上不像PD（D/E）XK核酸酶超家族的变体那样广泛或成功地分化或重新优化。这可能是由于放置在紧凑的HNH基序，这是同时参与蛋白质折叠，DNA结合和催化，以及使用平面，芳香族咪唑基团作为一般的基础上的多个空间限制。
Several unique protein folds that catalyze the hydrolysis of phosphodiester bonds have arisen independently in nature, including the PD(D/E)XK superfamily (typified by type II restriction endonucleases and many recombination and repair enzymes) and the HNH superfamily (found in an equally wide array of enzymes, including bacterial colicins and homing endonucleases). Whereas the identity and position of catalytic residues within the PD(D/E)XK superfamily are highly variable, the active sites of HNH nucleases are much more strongly conserved. In this study, the ability of an HNH nuclease to tolerate a mutation of its most conserved catalytic residue (its histidine general base), and the mechanism of the most active enzyme variant, were characterized. Conversion of this residue into several altered chemistries, glutamine, lysine, or glutamate, resulted in measurable activity. The histidine to glutamine mutant displays the highest residual activity and a pH profile similar to that of the wild-type enzyme. This activity is dependent on the presence of a neighboring imidazole ring, which has taken over as a less efficient general base for the reaction. This result implies that mutational pathways to alternative HNH-derived catalytic sites do exist but are not as extensively or successfully diverged or reoptimized in nature as variants of the PD(D/E)XK nuclease superfamily. This is possibly due to multiple steric constraints placed on the compact HNH motif, which is simultaneously involved in protein folding, DNA binding, and catalysis, as well as the use of a planar, aromatic imidazole group as a general base.