Alda-1调节线粒体醛脱氢酶（ALDH2）的动力学特性。,The FEBS Journal

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Alda-1调节线粒体醛脱氢酶（ALDH2）的动力学特性。
The FEBS Journal ( IF 5.5 ) Pub Date : 2016 Oct , DOI: 10.1111/febs.13833
Javier A. Belmont-Díaz ₁ , Belem Yoval-Sánchez ₁ , Luis F. Calleja-Castañeda ₁ , Juan P. Pardo Vázquez ₂ , José S. Rodríguez-Zavala ₁

Affiliation

线粒体醛脱氢酶（ALDH2）已被提出作为缺血再灌注过程中心脏保护的关键酶。该提议导致寻找ALDH2的活化剂，以开发保护心脏的药物。Alda-1是鉴定出的第一个ALDH2激活剂，其心脏保护作用已在体内得到广泛证明。但是，激活的机制还不完全清楚。晶体学研究表明，Alda-1与醛结合位点的入口结合。因此，Alda-1本质上应该是抑制剂。在本研究中，进行了动力学实验，以表征Alda-1对ALDH2的性质（动力学参数，限速步骤的确定，催化半胱氨酸的反应性）以及动力学机理（动力学类型，基材进入的顺序以及产品的释放）。结果表明，Alda-1极大地改变了ALDH2的性质，NAD +的Km降低了2.4倍，催化效率提高了4.4倍。但是，醛的Km增加了8.6倍，因此降低了催化效率。这些参数的变化导致行为复杂，其中Alda-1在低浓度的醛中起抑制剂作用，而在高浓度时起活化剂作用。此外，Alda-1与ALDH2的结合使脱酰作用的限制降低，并降低了催化半胱氨酸的pKa。最后，NADH抑制模式表明Alda-1诱导了底物进入和产物释放顺序的变化，这与两种底物都通过NAD +进入位点进入ALDH2的提议相一致。

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Alda-1 modulates the kinetic properties of mitochondrial aldehyde dehydrogenase (ALDH2).

Mitochondrial aldehyde dehydrogenase (ALDH2) has been proposed as a key enzyme in cardioprotection during ischemia-reperfusion processes. This proposal led to the search for activators of ALDH2 with the aim to develop cardioprotective drugs. Alda-1 was the first activator of ALDH2 identified and its cardioprotective effect has been extensively proven in vivo; however, the mechanism of activation is not fully understood. A crystallographic study showed that Alda-1 binds to the entrance of the aldehyde-binding site; therefore, Alda-1 should in essence be an inhibitor. In the present study, kinetic experiments were performed to characterize the effect of Alda-1 on the properties of ALDH2 (kinetic parameters, determination of the rate-limiting step, reactivity of the catalytic cysteine) and on the kinetic mechanism (type of kinetics, sequence of substrates entering, and products release). The results showed that Alda-1 dramatically modifies the properties of ALDH2, the Km for NAD+ decreased by 2.4-fold, and the catalytic efficiency increased 4.4-fold; however, the Km for the aldehyde increased 8.6-fold, thus, diminishing the catalytic efficiency. The alterations in these parameters resulted in a complex behavior, where Alda-1 acts as inhibitor at low concentrations of aldehyde and as an activator at high concentrations. Additionally, the binding of Alda-1 to ALDH2 made the deacylation less limiting and diminished the pKa of the catalytic cysteine. Finally, NADH inhibition patterns indicated that Alda-1 induced a change in the sequence of substrates entry and products release, in agreement with the proposal of both substrates entering ALDH2 by the NAD+ entrance site.

更新日期：2017-01-31

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