Investigations on the role of a solvent tunnel in the α-ketoglutarate dependent oxygenase factor inhibiting HIF (FIH)

Abstract

Non-heme Fe(II)/α-ketoglutarate (αKG)-dependent oxygenases catalyze a wide array of reactions through coupling oxidative decarboxylation of αKG to substrate oxygenation. This class of enzymes follows a sequential mechanism in which O reacts only after binding primary substrate, raising questions over how protein structure tailors molecular access to the Fe(II) cofactor. The enzyme "factor inhibiting hypoxia inducible factor" (FIH) senses pO in human cells by hydroxylating the C-terminal transactivation domain (CTAD), suggesting that structural elements limiting molecular access to the active site may limit the pO response. In this study, we tested the impact of a solvent-accessible tunnel in FIH on molecular access to the active site in FIH. The size of the tunnel was increased through alanine point mutagenesis (Y93A, E105A, and Q147A), followed by a suite of mechanistic and spectroscopic probes. Steady-state kinetics varying O or CTAD indicated that O passage through the tunnel was not affected by Ala substitutions, allowing us to conclude that this narrow tunnel did not impact pO sensing by FIH. Steady-state kinetics with varied αKG concentrations revealed increased substrate inhibition for the Ala variants, suggesting that a second αKG molecule may bind near the active site of FIH. If this solvent-accessible tunnel is the O entry tunnel, it may be narrow in order to permit O access while preventing metabolic intermediates, such as αKG, from inhibiting FIH under physiological conditions.

Evolution of tunnels in α/β-hydrolase fold proteins – what can we learn from studying epoxide hydrolases?

“…A detailed analysis is essential during the protein reengineering process. It can provide information about the residues blocking a potential new pathway, whose opening can promote significant modification of protein activity (52,67,68) or selectivity (23,29,69,70).…”

Protein Flexibility of the α-Ketoglutarate-Dependent Oxygenase Factor-Inhibiting HIF-1: Implications for Substrate Binding, Catalysis, and Regulation

“…Upon binding (M+αKG), the CTAD binding site shifts from low affinity (K D > 1 mM) to moderate affinity (K D = 80 μM), leading to a rigorously sequential binding order. 24 This has a practical effect, as there is but a narrow solvent accessible channel to the active site in the FIH/CTAD adduct, 14 which would prevent αKG from entering the active site were CTAD present. Otherwise, one could envision that CTAD binding to apoFIH might inhibit turnover by blocking αKG from reaching the active site.…”

“…In contrast, average B-factors in the refined structures show that thermal fluctuations of FIH decrease upon binding (M+αKG), 11,12 and a mechanistic study further revealed that the access of O 2 to the active site was not limited by constriction in a narrow channel. 14 These kinetic and structural data suggest that protein dynamics may be essential for ligand binding in FIH, as seen for ligand binding in other metalloenzymes. [15][16][17] Establishing the connection between protein dynamics and substrate binding may provide insight into the sequential chemical mechanism as well as providing hints for selectively targeting members of this important enzyme superfamily.…”

“…Structures of FIH bound to varied substrates and cofactor reveal that the equilibrium structure of FIH does not change when binding varied transition metals (M = Fe or Zn), αKG, CTAD, NO as a mimic of O 2 , or some combination thereof. − The use of a non-native metal permits structural studies while avoiding deleterious autoxidation reactions. In contrast, average B -factors in the refined structures show that thermal fluctuations of FIH decrease upon binding (M+αKG), , and a mechanistic study further revealed that the access of O 2 to the active site was not limited by constriction in a narrow channel . These kinetic and structural data suggest that protein dynamics may be essential for ligand binding in FIH, as seen for ligand binding in other metalloenzymes. − Establishing the connection between protein dynamics and substrate binding may provide insight into the sequential chemical mechanism as well as providing hints for selectively targeting members of this important enzyme superfamily. , …”

Inhibition of the Oxygen-Sensing Asparaginyl Hydroxylase Factor Inhibiting Hypoxia-Inducible Factor: A Potential Hypoxia Response Modulating Strategy

“…It is proposed that O 2 binds to an open coordination site on Fe­(II) (potentially induced by substrate binding as was precedented with other 2OG oxygenases , but notably not PHD2) yielding an Fe­(III) linked superoxide. The distal superoxide oxygen then attacks the C-2 carbonyl of 2OG, resulting in oxidative decarboxylation with formation of succinate, carbon dioxide (which likely efficiently leaves the active site), and a Fe­(IV)O intermediate, the latter of which is likely the active oxidant. ,, The ferryl species then stereospecifically abstracts a hydrogen atom from C-3 of HIF-α Asn803 to form a substrate radical and ferric hydroxide; a radical rebound process then gives the hydroxylated product and restores the Fe­(II) state. , The hydroxylated product and succinate are then released, , and the FIH-Fe­(II) complex can transition into a new catalytic cycle (Figure ).…”