类的机制我KDPG醛缩酶。
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富勒顿西南,格里菲斯JS,默克尔AB, Cheriyan M, Wymer新泽西,哈钦斯MJ, Fierke CA, Toone EJ,奈史密斯JH
类的机制我KDPG醛缩酶。
Bioorg医疗化学。2006年5月1日,14 (9):3002 - 10。2006年1月5 Epub。
- PubMed ID
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16403639 (在PubMed]
- 文摘
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体内,2-keto-3-deoxy-6-phosphogluconate (KDPG)醛缩酶催化可逆,立体定向retro-aldol乳沟KDPG丙酮酸和D-glyceraldehyde-3-phosphate。酶是一种lysine-dependent(一级)醛缩酶通过席夫碱独到的功能。在这里,我们提出一个基于晶体的这种酶机制研究野生型和突变体醛缩酶。的三维结构KDPG醛缩酶的嗜热生物Thermotoga maritima决心1.9。是标准的α/β桶结构观察类我醛缩酶。在活性位点赖氨酸我们观察清楚密度丙酮酸席夫碱。密度守恒的集群的硫酸盐离子结合残留物接近席夫碱也观察到。我们也决定的结构突变的大肠杆菌KDPG醛缩酶提出一般的酸/碱催化剂已被删除(E45N)。三聚物的一个亚基包含密度表明困丙酮酸carbinolamine中间。所有三个子单元包含一个磷酸离子有效绑定在一个位置相同的硫酸盐离子绑定在t . maritima酶。 The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.