On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis.
On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis.
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DOI：
10.1016/j.abb.2011.01.023
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发表时间：
2011-05-01
影响因子：
3.9
通讯作者：
Rhee KY
中科院分区：
文献类型：
--
作者：
de Carvalho LP;Ling Y;Shen C;Warren JD;Rhee KY
琥珀酸半醛脱氢酶（SSADH）是广泛存在的酶，其催化琥珀酸半醛（SSA）的NAD（P）+偶联氧化为琥珀酸，这是γ-氨基丁酸分流的最后一步。结核分枝杆菌编码两种旁系同源的SSADH（gabD 1和gabD 2）。在这里，我们描述的第一个机械特性的GabD 1，使用稳态动力学，pH速率曲线，1H-NMR，和动力学同位素效应。我们的研究结果证实SSA和NADP+作为底物，并表明，二价金属，如Mg 2+，线性化的时间过程。pH速率研究未能确定任何电离基团与pKa之间的5.5和10参与底物结合或限速化学。伯氘，溶剂和多种动力学同位素效应表明，亲核加成SSA是非常快的，其次是适度限速氢化物转移和快速硫酯水解。质子库存研究表明，一个单一的质子与溶剂敏感的限速步骤。总之，这些结果表明，产品解离和/或构象变化与它是限速的。使用人类同源酶的结构信息和1H-NMR，我们进一步确定亲核攻击发生在SSA的Si面，产生具有S立体化学的硫代半缩醛。氘化物转移到NADP+中的Pro-R位置产生硫酯中间体和[4A-2 H，4 B-1H] NADPH。提出了基于这些数据和结构信息的化学机制。
Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)+-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, 1H-NMR, and kinetic isotope effects. Our results confirmed SSA and NADP+ as substrates and demonstrated that a divalent metal, such as Mg2+, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pKa between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and 1H-NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP+ generates the thioester intermediate and [4A-2H, 4B-1H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.
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影响因子：
2.9
作者：
de Carvalho, Luiz Pedro S.;Blanchard, John S.
通讯作者：
Blanchard, John S.
影响因子：
2.9
作者：
Cohen-Gonsaud, M;Ducasse-Cabanot, S;Labesse, G
通讯作者：
Labesse, G
影响因子：
2.9
作者：
KIICK, DM;HARRIS, BG;COOK, PF
通讯作者：
COOK, PF
DOI：
10.1016/j.bbrc.2010.01.014
发表时间：
2010-01-29
影响因子：
3.1
作者：
Ahn, Jae-Woo;Kim, Yeon-Gil;Kim, Kyung-Jin
通讯作者：
Kim, Kyung-Jin
影响因子：
11.4
作者：
Kim, Yeon-Gil;Lee, Sujin;Kim, Kyung-Jin
通讯作者：
Kim, Kyung-Jin