Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase
Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase
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DOI：
10.1021/bi800212b
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发表时间：
2008-08-12
期刊：
影响因子：
2.9
通讯作者：
Sacchettini, James C.
中科院分区：
文献类型：
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作者：
Dey, Sanghamitra;Burton, Rodney L.;Sacchettini, James C.
结核分枝杆菌D-3-磷酸甘油酸脱氢酶的晶体结构已经用结合效应物L-丝氨酸和底物羟基丙酮酸磷酸分别以2.7和2.4埃的分辨率解析。亚基显示相同的极端不对称性，如在apostructur中看到的，并提供洞察丝氨酸结合和关闭的活性位点的模式。突变研究证实了参与丝氨酸结合的主要残基的身份，并表明环中包含导致亚基不对称的160度旋转位点的聚甘氨酸延伸可能在折叠中比在催化中发挥更大的作用。缺乏电子密度的辅因子，NADH，在任何晶体检查导致我们研究结合停流动力学分析。动力学数据表明，生产性的NADH结合，这将支持催化营业额，是依赖于底物的存在。这一观察结果，沿着与底物在活性位点的结合，但在非生产性构象，表明一种可能的机制，其中底物的初始结合导致与辅因子的相互作用增强，伴随着催化关键残基侧链的重排。此外，与具有辅因子和底物类似物的人D-3-磷酸甘油酸脱氢酶的截短形式的结构的比较，提供了对催化过程中发生的构象变化的洞察。
The crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase has been solved with bound effector, L-serine, and substrate, hydroxypyruvic acid phosphate, at resolutions of 2.7 and 2.4 angstrom, respectively. The subunits display the same extreme asymmetry as seen in the apostructure and provide insight into the mode of serine binding and closure of the active site. Mutagenesis studies confirm the identity of the main residues involved in serine binding and suggest that the poly glycine stretch in the loop that contains the locus for the 160 degrees rotation that leads to subunit asymmetry may have a larger role in folding than in catalysis. The lack of electron density for the cofactor, NADH, in any of the crystals examined led us to study binding by stopped flow kinetic analysis. The kinetic data suggest that productive NADH binding, that would support catalytic turnover, is dependent on the presence of substrate. This observation, along with the binding of substrate in the active site, but in an unproductive conformation, suggests a possible mechanism where initial binding of substrate leads to enhanced interaction with cofactor accompanied by a rearrangement of catalytically critical residue side chains. Furthermore, comparison to the structure of a truncated form of human D-3-phosphoglycerate dehydrogenase with cofactor and a substrate analog, provides insight into the conformational changes that occur during catalysis.