Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction.
Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction.
复制标题
GDP-甘露糖甘露糖基水解酶反应中解离机制的突变、结构和动力学证据。
DOI：
10.1021/bi050583v
复制
发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Mildvan,AlbertS
中科院分区：
文献类型：
--
作者：
Xia,Zuyong;Azurmendi,HugoF;Lairson,LukeL;Withers,StephenG;Gabelli,SandraB;Bianchet,MarioA;Amzel,LMario;Mildvan,AlbertS
GDP-甘露糖水解酶（GDPMH）通过亲核取代并在糖的异头C1原子处转化来催化GDP-α-d-糖的水解，一般通过H124进行碱催化。三条线索的证据表明一个机制与分离的字符。首先，在GDPMH− Mg 2 +−GDP·Tris+络合物的1.3 <$X射线结构中[Gabelli，S. B.，等人（2004）Structure 12，927 - 935]，GDP离去基团与五种催化组分相互作用：R37、Y103、R52、R65和必需的Mg 2+。根据位点特异性突变体onkcat的作用确定，这些组分对催化作用的贡献因子分别为24、100、309、24和≥105倍。R37和Y103都与GDP的β-磷酸结合，并且相距仅5.0 nm。因此，R37 Q/Y103 F双突变体对Km的影响表现为两个单突变体对Km的部分加和效应，表明R37和Y103在促进催化方面具有协同效应，而对Km的影响表现为拮抗效应。第二，保守残基D22的位置可以接受来自C1位发生取代的糖的C2−OH基团的氢键，如通过将α-d-甘露糖基模拟到糖结合位点所示。D22 A和D22 N突变使GDP-α-d-甘露糖水解的kcat分别降低了102.1和102.6倍，对Km的影响较小，表明D22阴离子稳定了阳离子氧碳正离子过渡态。第三，氟化底物GDP-2F-α-d-甘露糖，其阳离子氧碳正离子过渡态将因电子撤回而不稳定，表现出16倍的inccat降低和较小的2.5倍的Km增加。D22 A和D22 N突变进一步降低了GDP-2F-α-d-甘露糖的kcat值，与GDP-α-d-甘露糖相似，并降低了氟化底物的Km值。选择组氨酸作为一般碱基而不是谷氨酸（谷氨酸是其他营养素酶中的首选碱基），并不是因为组氨酸的碱性更强，因为活性复合物中E124的pKa（7.7）超过了H124的pKa（6.7），H124 E突变显示在pH 9.3时，K降低了102.2倍，K增加了4.0倍。类似地，在X射线结构中检测到的催化三联体（H124-Y127-P120）对于定向H124是不必要的，因为Y127 F突变对于以H124或E124作为总碱基的kcat和Km只有2倍的影响。因此，中性组氨酸而不是阴离子谷氨酸盐可能是必要的，以保持活性复合物中的电中性。
GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-α-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 Å X-ray structure of the GDPMH−Mg2+−GDP·Tris+complex [Gabelli, S. B., et al. (2004)Structure 12, 927−935], the GDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essential Mg2+. As determined by the effects of site-specific mutants onkcat, these components contribute factors of 24-, 100-, 309-, 24-, and ≥105-fold, respectively, to catalysis. Both R37 and Y103 bind the β-phosphate of GDP and are only 5.0 Å apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additive effects of the two single mutants onkcat, indicating cooperativity of R37 and Y103 in promoting catalysis, and antagonistic effects onKm. Second, the conserved residue, D22, is positioned to accept a hydrogen bond from the C2−OH group of the sugar undergoing substitution at C1, as was shown by modeling an α-d-mannosyl group into the sugar binding site. The D22A and D22N mutations decreasedkcatby factors of 102.1and 102.6, respectively, for the hydrolysis of GDP-α-d-mannose, and showed smaller effects onKm, suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinated substrate, GDP-2F-α-d-mannose, for which a cationic oxocarbenium transition state would be destabilized by electron withdrawal, exhibited a 16-fold decrease inkcatand a smaller, 2.5-fold increase inKm. The D22A and D22N mutations further decreased thekcatwith GDP-2F-α-d-mannose to values similar to those found with GDP-α-d-mannose, anddecreasedtheKmof the fluorinated substrate. The choice of histidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to the greater basicity of histidine, since the pKaof E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 102.2-folddecreaseinkcatand a 4.0-fold increase inKmat pH 9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessary for orienting H124, since the Y127F mutation had only 2-fold effects onkcatandKmwith either H124 or E124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessary to preserve electroneutrality in the active complex.