Influence of -CHNH Substitution in C8-CoA on the Kinet

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• 作者：Karen M. Peterson ; K. V. Gopalan ; and D. K. Srivastava
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：October 17, 2000
• 年：2000
• 卷：39
• 期：41
• 页码：12659 - 12670
• 全文大小：153K
• 年卷期：v.39,no.41(October 17, 2000)
• ISSN：1520-4995

文摘

We previously reported that the kinetic profiles for the association and dissociation offunctionally diverse C₈-CoA-ligands, viz., octanoyl-CoA (substrate), octenoyl-CoA (product), and octynoyl-CoA (inactivator) with medium chain acyl-CoA dehydrogenase (MCAD), were essentially identical,suggesting that the protein conformational changes played an essential role during ligand binding and/orcatalysis [Peterson, K. L., Sergienko, E. E., Wu, Y., Kumar, N. R., Strauss, A. W., Oleson, A. E., Muhonen,W. W., Shabb, J. B., and Srivastava, D. K. (1995) Biochemisry 34, 14942-14953]. To ascertain thestructural basis of the above similarity, we investigated the kinetics of association and dissociation ofimages/gifchars/alpha.gif" BORDER=0>-CHimages/entities/rarr.gif">NH-substituted C₈-CoA, namely, 2-azaoctanoyl-CoA, with the recombinant form of human liverMCAD. The rapid-scanning and single wavelength stopped-flow data for the binding of 2-azaoctanoyl-CoA to MCAD revealed that the overall interaction proceeds via two steps. The first (fast) step involvesthe formation of an enzyme-ligand collision complex (with a dissociation constant of K_c), followed bya slow isomerization step (with forward and reverse rate constants of k_f and k_r, respectively) withconcomitant changes in the electronic structure of the enzyme-bound FAD. Since the latter step involvesa concurrent change in the enzyme's tryptophan fluorescence, it is suggested that the isomerization stepis coupled to the changes in the protein conformation. Although the overall binding affinity (K_d) of theenzyme-2-azaoctanoyl-CoA complex is similar to that of the enzyme-octenoyl-CoA complex, theirmicroscopic equilibria within the collision and isomerized complexes show an opposite relationship. Theseresults coupled with the isothermal titration microcalorimetric studies lead to the suggestion that theelectrostatic interaction within the enzyme site phase modulates the microscopic steps, as well as theircorresponding ground and transition states, during the course of the enzyme-ligand interaction.