NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity

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• 作者：Arthur G. Roberts ; M. Dolores Dí ; az ; Jed N. Lampe ; Laura M. Shireman ; Jeffrey S. Grinstead ; Michael J. Dabrowski ; Josh T. Pearson ; Michael K. Bowman ; William M. Atkins ; and A. Patricia Campbell
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：February 14, 2006
• 年：2006
• 卷：45
• 期：6
• 页码：1673 - 1684
• 全文大小：286K
• 年卷期：v.45,no.6(February 14, 2006)
• ISSN：1520-4995

文摘

Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins.Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropiccooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery arestill not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR)spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450_eryF.UV/vis absorbance spectra of P450_eryF demonstrated that binding occurs with apparent negative homotropiccooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-deriveddissociation constants of K_S = 4 and 200 s/entities/mgr.gif">M, respectively. The broadening and shifting observed in the2D-{¹H,¹⁵N}-HSQC-monitored titrations of ¹⁵N-Phe-labeled P450_eryF with 9-AP and TST indicated bindingon intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hill-equation-derived K_S values for these two ligands. Regardless of the type of spectral perturbation observed(broadening for 9-AP and shifting for TST), the ¹⁵N-Phe NMR resonances most affected were the samein each titration, suggesting that the two ligands "contact" the same phenylalanines within the active siteof P450_eryF. This finding is in agreement with X-ray crystal structures of bound P450_eryF showing differentligands occupying similar active-site niches. Complex spectral behavior was additionally observed for asmall collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450_eryF conformational substates. A structural and energeticmodel is presented that combines the energetics and structural aspects of 9-AP and TST binding derivedfrom these observations.