Unveiling the Catalytic Mechanism of NADP+-Dependent Isocitrate Dehydrogenase with QM/MM Calculations

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• 作者：Rui P. P. Neves ; Pedro A. Fernandes ; Maria J. Ramos
• 刊名：ACS Catalysis
• 出版年：2016
• 出版时间：January 4, 2016
• 年：2016
• 卷：6
• 期：1
• 页码：357-368
• 全文大小：687K
• ISSN：2155-5435

文摘

We have determined the catalytic mechanism of the human cytosolic homodimeric isocitrate dehydrogenase (hICDH), an enzyme involved in the regulation of tumorogenesis. Our study constitutes the first theoretical attempt to describe the entire catalytic cycle of hICDH. In agreement with earlier experimental proposals, the catalysis was shown to proceed in three steps: (1) NADP⁺ reduction by the isocitrate substrate with the help of the Lys212^B base, (2) β-decarboxylation of the resulting oxalosuccinate, generating an enolate, and (3) protonation of this intermediate by Tyr139^A, giving rise to the α-ketoglutarate product. Our study supports that the β-decarboxylation of oxalosuccinate is the most likely rate-limiting step, with an activation Gibbs free energy of 16.5 kcal mol^–1. The calculated values are in close agreement with the 16–17 kcal mol^–1 range, derived by the application of transition state theory to the reaction rates determined experimentally (11 to 38 s^–1). We emphasize the role of Mg²⁺ and Asp275^A, whose acid/base properties throughout the catalytic cycle were found to lower the barrier to physiologically competent values. Aside from its chemical dual role (as a base, deprotonating Lys212^B, and as an acid, protonating the basic Tyr139^A deprotonated by the enolate intermediate), Asp275^A also establishes hydrogen bonds with Arg132^A and Tyr 139^A that become shorter at critical transition states. These residues were shown to influence both the rate and the efficiency of hICDH. The knowledge drawn in this study provides new insights into future clinical and bioengineering applications of hICDH: namely, in the development of techniques to regulate the growth of glioblastomas and to capture and store carbon dioxide. Moreover, it further extends the comprehension of (1) the hydrogen/charge transfer mechanism that regulates the hydrogenation of NADP⁺ to NADPH, an ubiquitous biochemical reaction, and (2) the role of divalent metals as key structure elements in the family of NAD(P)⁺-dependent β-decarboxylases.