Backbone flexibility, conformational change, and catalysis in a phosphohexomutase from Pseudomonas aeruginosa
Backbone flexibility, conformational change, and catalysis in a phosphohexomutase from Pseudomonas aeruginosa
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DOI：
10.1021/bi8005219
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发表时间：
2008-09-02
期刊：
影响因子：
2.9
通讯作者：
Beamer, Lesa J.
中科院分区：
文献类型：
--
作者：
Schramm, Andrew M.;Mehra-Chaudhary, Ritcha;Beamer, Lesa J.
铜绿假单胞菌的磷酸甘露变位酶/磷酸葡萄糖变位酶（PMM/PGM）参与几种复合碳水化合物的生物合成，包括藻酸盐、脂多糖和鼠李糖脂。以前的结构研究表明，这种蛋白质的底物的结合产生的C-末端结构域的旋转，改变活性位点从一个开放的裂缝中的脱辅基酶到一个深，溶剂难以接近的口袋中发生磷酰基转移。我们在此报告定点诱变，动力学和结构研究，在检查域3和4之间的铰链中的残基的作用，以及残基参与酶-底物接触，并帮助形成多域的活性位点的“盖子”。我们发现铰链区残基的骨架柔性（例如，脯氨酸突变为甘氨酸/丙氨酸）影响反应效率，kat降低约10倍，K-m增加约2倍。此外，热力学分析表明，这些变化主要是由于熵效应，与多肽骨架的柔性增加一致，导致形成催化生产活性位点的可能性降低。铰链残基的这些结果与酶活性位点突变体的结果形成对比，后者对酶动力学具有深远的影响（k（cat）/K-m降低10（2）-10（3）倍），并且还显示其热力学参数相对于野生型（WT）酶的热力学参数存在实质性差异。这些研究支持蛋白质铰链中的多肽柔性可以进化以优化和调节反应速率的概念。
The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from the bacterium Pseudomonas aeruginosa is involved in the biosynthesis of several complex carbohydrates, including alginate, lipopolysaccharide, and rhamnolipid. Previous structural studies of this protein have shown that binding of substrates produces a rotation of the C-terminal domain, changing the active site from an open cleft in the apoenzyme into a deep, solvent inaccessible pocket where phosphoryl transfer takes place. We report herein site-directed mutagenesis, kinetic, and structural studies in examining the role of residues in the hinge between domains 3 and 4, as well as residues that participate in enzyme-substrate contacts and help form the multidomain "lid" of the active site. We find that the backbone flexibility of residues in the hinge region (e.g., mutation of proline to glycine/alanine) affects the efficiency of the reaction, decreasing kat by similar to 10-fold and increasing K-m by similar to 2-fold. Moreover, thermodynamic analyses show that these changes are due primarily to entropic effects, consistent with an increase in the flexibility of the polypeptide backbone leading to a decreased probability of forming a catalytically productive active site. These results for the hinge residues contrast with those for mutants in the active site of the enzyme, which have profound effects on enzyme kinetics (10(2)-10(3)-fold decrease in k(cat)/K-m) and also show substantial differences in their thermodynamic parameters relative to those of the wild-type (WT) enzyme. These studies support the concept that polypeptide flexibility in protein hinges may evolve to optimize and tune reaction rates.