Thermodynamics of Ligand Binding and Catalysis in Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Comparative Studies Involving Normal and 3'-Dephosphorylated C8-CoAs and Wild-Type and Asn

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• 作者：Kevin L. Peterson ; Karen M. Peterson ; and D. K. Srivastava
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 8, 1998
• 年：1998
• 卷：37
• 期：36
• 页码：12659 - 12671
• 全文大小：263K
• 年卷期：v.37,no.36(September 8, 1998)
• ISSN：1520-4995

文摘

Following our demonstration that the terminal 3'-phosphate group of acyl-CoA substrates (whichis 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 becameinterested in delineating its thermodynamic contribution in stabilizing the "ground" and "transition" statestructures during enzyme catalysis. Since the 3'-phosphate group of the coenzyme A thiolester has thepotential to form a hydrogen bond with the side chain group of Asn-191, these studies were performedutilizing both normal and 3'-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulativelyreferred to as C₈-CoA) as the physiological substrate and product of the enzyme, respectively, as well asutilizing wild-type and Asn191 images/entities/rarr.gif"> Ala (N191A) site-specific mutant enzymes. The experimental datarevealed that the enthalpic contribution of the 3'-phosphate group was similar in both ground and transitionstates, and was primarily derived from the London-van der Waals interactions (between the 3'-phosphategroup of C₈-CoA and the surrounding protein moiety), rather than from the potential hydrogen bonding.The temperature dependence of images/gifchars/Delta.gif" BORDER=0 >Himages/entities/deg.gif"> for the binding of octenoyl-CoA and 3'-dephosphooctenoyl-CoArevealed that the deletion of the 3'-phosphate group from octenoyl-CoA increased the magnitude of theheat capacity changes (images/gifchars/Delta.gif" BORDER=0 >C_pimages/entities/deg.gif">) from -0.53 to -0.59 kcal mol^-1 K^-1. Although the latter effect could beattributed to an increase in the relative hydrophobicity of the ligand, the experimentally observed images/gifchars/Delta.gif" BORDER=0 >C_pimages/entities/deg.gif">'s(for either of the ligands) could not be predicted on the basis of the changes in the solvent-accessiblesurface areas of the enzyme and ligand species. These coupled with the fact that the images/gifchars/Delta.gif" BORDER=0 >C_pimages/entities/deg.gif"> for the bindingof octenoyl-CoA to pig kidney MCAD (which is believed to be structurally identical to human liverMCAD) 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 images/gifchars/Delta.gif" BORDER=0 >C_pimages/entities/deg.gif"> values of theenzyme-ligand complexes from their X-ray crystallographic data. Arguments are presented that certainintrinisic limitations of the crystallographic data preclude kinetic and thermodynamic predictions aboutthe enzyme-ligand complexes and enzyme catalysis.