Peterson K L, Peterson K M, Srivastava D K
Biochemistry Department, North Dakota State University, Fargo 58105, USA.
Biochemistry. 1998 Sep 8;37(36):12659-71. doi: 10.1021/bi980949m.
Following our demonstration that the terminal 3'-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751-760], we became interested in delineating its thermodynamic contribution in stabilizing the "ground" and "transition" state structures during enzyme catalysis. Since the 3'-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3'-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 --> Ala (N191A) site-specific mutant enzymes. The experimental data revealed that the enthalpic contribution of the 3'-phosphate group was similar in both ground and transition states, and was primarily derived from the London-van der Waals interactions (between the 3'-phosphate group of C8-CoA and the surrounding protein moiety), rather than from the potential hydrogen bonding. The temperature dependence of DeltaH degrees for the binding of octenoyl-CoA and 3'-dephosphooctenoyl-CoA revealed that the deletion of the 3'-phosphate group from octenoyl-CoA increased the magnitude of the heat capacity changes (DeltaCp degrees) from -0.53 to -0.59 kcal mol-1 K-1. Although the latter effect could be attributed to an increase in the relative hydrophobicity of the ligand, the experimentally observed DeltaCp degrees's (for either of the ligands) could not be predicted on the basis of the changes in the solvent-accessible surface areas of the enzyme and ligand species. These coupled with the fact that the DeltaCp degrees for the binding of octenoyl-CoA to pig kidney MCAD (which is believed to be structurally identical to human liver MCAD) is only -0.37 kcal mol-1 K-1 [Srivastava, D. K., Wang, S., and Peterson, K. L. (1997) Biochemistry 36, 6359-6366] prompt us to question the reliability of predicting the DeltaCp degrees values of the enzyme-ligand complexes from their X-ray crystallographic data. Arguments are presented that certain intrinisic limitations of the crystallographic data preclude kinetic and thermodynamic predictions about the enzyme-ligand complexes and enzyme catalysis.
在我们证明酰基辅酶A底物的3'-磷酸末端基团（该基团位于蛋白质结构外部，完全暴露于外部溶剂环境）在重组人肝中链酰基辅酶A脱氢酶（MCAD）催化的反应中发挥功能作用之后[彼得森，K. L.，和斯里瓦斯塔瓦，D. K.（1997年）《生物化学杂志》325卷，751 - 760页]，我们开始对描绘其在酶催化过程中稳定“基态”和“过渡态”结构的热力学贡献感兴趣。由于辅酶A硫酯的3'-磷酸基团有可能与Asn - 191的侧链基团形成氢键，这些研究分别使用辛酰辅酶A和辛烯酰辅酶A（统称为C8 - CoA）的正常形式和3'-去磷酸化形式作为该酶的生理底物和产物，以及使用野生型和Asn191→Ala（N191A）位点特异性突变酶来进行。实验数据表明，3'-磷酸基团在基态和过渡态中的焓贡献相似，并且主要源自伦敦 - 范德华相互作用（C8 - CoA的3'-磷酸基团与周围蛋白质部分之间），而非潜在的氢键作用。辛烯酰辅酶A和3'-去磷酸辛烯酰辅酶A结合的ΔH°对温度的依赖性表明，从辛烯酰辅酶A中去除3'-磷酸基团会使热容变化幅度（ΔCp°）从 - 0.53增加到 - 0.59千卡·摩尔⁻¹·K⁻¹。尽管后一种效应可归因于配体相对疏水性的增加，但根据酶和配体物种可溶剂化表面积的变化无法预测实验观察到的（对于任何一种配体的）ΔCp°。这些情况再加上辛烯酰辅酶A与猪肾MCAD（据信其结构与人类肝MCAD相同）结合的ΔCp°仅为 - 0.37千卡·摩尔⁻¹·K⁻¹[斯里瓦斯塔瓦，D. K.，王，S.，和彼得森，K. L.（1997年）《生物化学》36卷，6359 - 6366页]，促使我们质疑从X射线晶体学数据预测酶 - 配体复合物的ΔCp°值的可靠性。文中提出论据表明，晶体学数据的某些内在局限性妨碍了对酶 - 配体复合物和酶催化的动力学和热力学预测。
