突变、结构和动力学的证据的离解机理GDP-mannose mannosyl水解酶反应。
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夏Z, Azurmendi高频Lairson LL,威瑟斯SG,加百利某人,Bianchet MA Amzel LM, Mildvan
突变、结构和动力学的证据的离解机理GDP-mannose mannosyl水解酶反应。
生物化学。2005年6月28日,44 (25):8989 - 97。
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15966723 (在PubMed]
- 文摘
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GDP-mannose水解酶(GDPMH)催化的水解GDP-alpha-d-sugars通过亲核取代反转异头C1原子的糖,与一般由H124碱催化。三行证据表明一种机制与离解的性格。首先,在的1.3 x射线结构GDPMH-Mg (2 +) -GDP.Tris(+)复杂[加百利,s B。12,et al。(2004)结构,927 - 935],GDP离去基团与五催化组件:R37, Y103 R52, R65,重要毫克(2 +)。由特定站点的影响突变体在k (cat),这些组件的贡献因素24 - 100 - 309 - 24 - > / = 10(5)倍,分别催化。R37和Y103绑定GDP的beta-phosphate相距仅5.0。因此,R37Q / Y103F双突变体展览部分添加剂的影响两个单突变体在k (cat),指示协同R37 Y103促进催化,对k (m)和敌对的影响。D22摊位,第二,守恒的残留物是能够接受氢键接受替代糖C2-OH集团的C1,建模一个alpha-d-mannosyl组就是一个说明糖结合位点。D22A和D22N突变因素降低k (cat)的10(2.1)和(2.6),分别为GDP-alpha-d-mannose的水解,对k (m)和显示小的影响,这表明D22摊位阴离子稳定阳离子oxocarbenium过渡状态。GDP-2F-alpha-d-mannose,第三,氟化衬底的阳离子oxocarbenium过渡状态将由电子不稳定,表现出16倍降低k(猫)和一个更小的,2.5倍增加k (m)。 The D22A and D22N mutations further decreased the k(cat) with GDP-2F-alpha-d-mannose to values similar to those found with GDP-alpha-d-mannose, and decreased the K(m) of 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 pK(a) of E124 in the active complex (7.7) exceeded that of H124 (6.7), and the H124E mutation showed a 10(2.2)-fold decrease in k(cat) and a 4.0-fold increase in K(m) at 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 on k(cat) and K(m) with 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.