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Inverse Solvent Isotope Effects Arising from Substrate Triggering in the Factor Inhibiting Hypoxia Inducible Factor

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journal contribution
posted on 2013-03-05, 00:00 authored by John A. Hangasky, Evren Saban, Michael J. Knapp
Oxygen homeostasis plays a critical role in angiogenesis, erythropoiesis, and cell metabolism. Oxygen homeostasis is set by the hypoxia inducible factor-1α (HIF-1α) pathway, which is controlled by factor inhibiting HIF-1α (FIH). FIH is a non-heme Fe­(II), α-ketoglutarate (αKG)-dependent dioxygenase that inhibits HIF-1α by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1α at HIF-Asn803. A tight coupling between CTAD binding and O2 activation is essential for hypoxia sensing, making changes in the coordination geometry of Fe­(II) upon CTAD encounter a crucial feature of this enzyme. Although the consensus chemical mechanism for FIH proposes that CTAD binding triggers O2 activation by causing the Fe­(II) cofactor to release an aquo ligand, experimental evidence of this has been absent. More broadly, this proposed coordination change at Fe­(II) has not been observed during steady-state turnover in any αKG oxygenase to date. In this work, solvent isotope effects (SIEs) were used as a direct mechanistic probe of substrate-triggered aquo release in FIH, as inverse SIEs (SIE < 1) are signatures for pre-equilibrium aquo release from metal ions. Our mechanistic studies of FIH have revealed inverse solvent isotope effects in the steady-state rate constants at limiting concentrations of CTAD or αKG [D2Okcat/KM(CTAD) = 0.40 ± 0.07, and D2Okcat/KM(αKG) = 0.32 ± 0.08], providing direct evidence of aquo release during steady-state turnover. Furthermore, the SIE at saturating concentrations of CTAD and αKG was inverse (D2Okcat = 0.51 ± 0.07), indicating that aquo release occurs after CTAD binds. The inverse kinetic SIEs observed in the steady state for FIH can be explained by a strong Fe–OH2 bond. The stable Fe–OH2 bond plays an important part in FIH’s regulatory role over O2 homeostasis in humans and points toward a strategy for tightly coupling O2 activation with CTAD hydroxylation that relies on substrate triggering.

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