Kinetic control of thiamin diphosphate activation in enzymes studied by proton-nitrogen correlated NMR spectroscopy.
Kinetic control of thiamin diphosphate activation in enzymes studied by proton-nitrogen correlated NMR spectroscopy.
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通过质子氮相关核磁共振波谱研究酶中二磷酸硫胺素活化的动力学控制。
DOI：
10.1021/bi050522x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Kern,Dorothee
中科院分区：
文献类型：
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作者：
Tittmann,Kai;Neef,Holger;Golbik,Ralph;Hubner,Gerhard;Kern,Dorothee
对硫胺素二磷酸进行了质子-氮相关 NMR 研究，硫胺素二磷酸在 4'-氨基处用 15N 进行了专门标记。用来自运动发酵单胞菌的野生型丙酮酸脱羧酶和 E50Q 变体的脱辅基酶重建标记辅酶后，与游离辅酶相比，在 pH 5.9 时观察到 15 N 信号的高场位移~4 ppm，表明在 4'-氨基氮处有更高的电子密度。 酶结合状态。 1 H−15 N 异核单量子相干NMR谱中15 N信号的化学位移的pH依赖性揭示了具有几乎相同的化学位移终点的游离辅酶和重构辅酶的典型滴定曲线。游离辅酶和酶结合辅酶的转变中点分别为 pH 5.3 和 5.0。我们得出的结论是，ThDP 酶中 C2-H 去质子化速率的巨大加速主要是由于活性位点中辅因子的强制 V 构象非常适合分子内酸碱催化。
Proton−nitrogen correlated NMR studies were performed on thiamin diphosphate, which has been specifically labeled with15N at the 4‘-amino group. After reconstitution of the labeled coenzyme with the apoenzymes of both wild-type pyruvate decarboxylase fromZymomonas mobilisand the E50Q variant, a high-field shift of the15N signal of ∼4 ppm is observed at pH 5.9 when compared to that of the free coenzyme, indicating a higher electron density at the 4‘-amino nitrogen in the enzyme-bound state. The pH dependence of the chemical shift of the15N signals in the1H−15N heteronuclear single-quantum coherence NMR spectra reveals typical titration curves for the free as well as the reconstituted coenzyme with nearly identical chemical shift end points. The midpoints of the transitions are at pH 5.3 and 5.0 for the free and enzyme-bound coenzyme, respectively. We conclude that the tremendous rate acceleration of C2-H deprotonation in ThDP enzymes is mainly the result of the enforcedVconformation of the cofactor in the active site being perfectly suited to allowing intramolecular acid−base catalysis.