Inverse Solvent Isotope Effects Demonstrate Slow Aquo Release from Hypoxia Inducible Factor-Prolyl Hydroxylase (PHD2)

Abstract

Prolyl Hydroxylase Domain 2 (PHD2) is deemed a primary oxygen sensor in humans yet many details of its underlying mechanism are still not fully understood. (Fe2++αKG)PHD2 is 6-coordinate, with a 2His/1Asp facial triad occupying 3 coordination sites, a bidentate α-ketoglutarate occupying two sites and an aquo ligand in the final site. Turnover is thought to be initiated upon release of the aquo ligand, creating a site for O2 to bind at the iron. Herein we show that steady-state turnover is faster under acidic conditions, with kcat exhibiting a kinetic pKa = 7.22. A variety of spectroscopic probes were employed to identify the active-site acid, through comparison of (Fe2++αKG)PHD2 at pH 6.50 with pH 8.50. The near-UV circular dichroism spectrum was virtually unchanged at elevated pH, indicating that the secondary structure did not change as a function of pH. UV-visible and Fe X-ray absorption spectroscopy indicated that the primary coordination sphere of Fe2+ changed upon increasing the pH; EXAFS analysis found a short Fe-(O/N) bond length of 1.96 Å at pH 8.50, strongly suggesting that the aquo ligand was deprotonated at this pH. Solvent isotope effects were measured during steady-sate turnover over a wide pH-range, with an inverse SIE of on kcat observed (D2Okcat = 0.91 ± 0.03) for the acid form; a similar SIE was observed for the basic form of enzyme (D2Okcat = 0.9 ± 0.1), with an acid equilibrium offset of ΔpKa = 0.67 ± 0.04. The inverse SIE indicated that aquo release from the active site Fe2+ immediately precedes a rate-limiting step, suggesting that turnover in this enzyme may be partially limited by the rate of O2 binding or activation, and suggesting that aquo release is relatively slow. The unusual kinetic pKa further suggested that PHD2 might function physiologically to sense both intracellular pO2 as well as pH, which could provide for feedback between anaerobic metabolism and hypoxia sensing.

Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change

“…Although the rate-limiting step for PHD2 turnover remains to be identified, this step precedes decarboxylation when ODD is saturating, as no oxidized intermediates were observed in the pre-steady state [34], and a slightly inverse solvent isotope effect on k cat was observed [23]. The results reported herein suggest that the kinetically coupled closing of the β2β3 loop may limit the rate of turnover under conditions of low ODD concentration.…”

“…Recombinant human PHD2 (corresponding to residues 178–426 of the full-length PHD2 sequence) was expressed and purified as previously reported [23]. PHD2 was expressed with an N-terminal GST tag (GST-PHD2) in E. coli BL21(DE3) cells using a pGEX-4T-1 vector (Stratagene).…”

“…The large variation in reported values for the Michaelis constants, with K M(NODD) values ranging from 20 – 50 μM and K M(CODD) values ranging from 1 – 30 μM [17, 19, 23], is largely a reflection of three factors that introduce variation into the kinetic data, two of which are common to the broad class of 2OG oxygenases. First is the inherent difficulty of measuring turnover for the 2OG oxygenases, due to the relatively slow rate of turnover and the absence of chromophoric substrates.…”

“…This in turn greatly impacts assays that rely on the rate of CO 2 liberation or O 2 consumption, and certainly leads to over-estimation of turnover rates. Third is the pK A = 7.2 of the Fe 2+ -OH 2 group in (Fe+2OG)PHD2 [23], leading to a significant decrease in activity as pH is increased above the p K a [27]. Based on these divergent Michaelis constants, and the potential for uncoupled oxidative decarboxylation to inflate the observed rates, we felt that the substrate preference and rates could be better defined by directly measuring the formation of hydroxylated product (ODD OH ) using MALDI-TOF [28, 29].…”

Oxygen sensing strategies in mammals and bacteria

“…Pre-steady state kinetics indicated that PHD2 reacted with O 2 very slowly when compared to other αKG oxygenases, and related freeze-quench experiments failed to isolate the putative ferryl intermediate [77], which was consistent with O 2 -activation as the rate-limiting step in PHD2. Steady-state kinetics further supports rate-limiting O 2 -activation in PHD2 [78]. In order for the HIF hydroxylases to be good O 2 sensors, O 2 binding or a step subsequent to O 2 binding should be rate limiting as this would lead to proportionate changes in activity with pO 2 .…”