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.

Two primary O2-sensors for humans are the HIF-hydroxylases, enzymes that hydroxylate specific residues of the hypoxia inducible factor-α (HIF). These enzymes are factor inhibiting HIF (FIH) and prolyl hydroxylase-2 (PHD2), each an α-ketoglutarate (αKG) dependent, nonheme Fe(II) dioxygenase. Although the two enzymes have similar active sites, FIH hydroxylates Asn803 of HIF-1α while PHD2 hydroxylates Pro402 and/or Pro564 of HIF-1α. The similar structures but unique functions of FIH and PHD2 make them prime targets for selective inhibition leading to regulatory control of diseases such as cancer and stroke. Three classes of iron chelators were tested as inhibitors for FIH and PHD2: pyridines, hydroxypyrones/hydroxypyridinones and catechols. An initial screen of the ten small molecule inhibitors at varied [αKG] revealed a non-overlapping set of inhibitors for PHD2 and FIH. Dose response curves at moderate [αKG] ([αKG] ~ KM) showed that the hydroxypyrones/hydroxypyridinones were selective inhibitors, with IC50 in the µM range, and that the catechols were generally strong inhibitors of both FIH and PHD2, with IC50 in the low µM range. As support for binding at the active site of each enzyme as the mode of inhibition, electron paramagnetic resonance (EPR) spectroscopy were used to demonstrate inhibitor binding to the metal center of each enzyme. This work shows some selective inhibition between FIH and PHD2, primarily through the use of simple aromatic or pseudo-aromatic chelators, and suggests that hydroxypyrones and hydroxypyridones may be promising chelates for FIH or PHD2 inhibition.

The factor inhibiting HIF (FIH) is one of the primary oxygen sensors in human cells, controlling gene expression by hydroxylating the α-subunit of the hypoxia inducible transcription factor (HIF). As FIH is an alpha-ketoglutarate dependent non-heme iron dioxygenase, oxygen activation is thought to precede substrate hydroxylation. The coupling between oxygen activation and substrate hydroxylation was hypothesized to be very tight, in order for FIH to fulfill its function as a regulatory enzyme. Coupling was investigated by looking for reactive oxygen species production during turnover. We used alkylsulfatase (AtsK), a metabolic bacterial enzyme with a related mechanism and similar turnover frequency, for comparison, and tested both FIH and AtsK for H2O2, O2− and OH• formation under steady and substrate-depleted conditions. Coupling ratios were determined by comparing the ratio of substrate consumed to product formed. We found that AtsK reacted with O2 on the seconds timescale in the absence of prime substrate, and uncoupled during turnover to produce H2O2; neither O2− nor OH• were detected. In contrast, FIH was unreactive toward O2 on the minutes timescale in the absence of prime substrate, and tightly coupled during steady-state turnover; we were unable to detect any reactive oxygen species produced by FIH. We also investigated the inactivation mechanisms of these enzymes and found that AtsK likely inactivated due to deoligomerizion, whereas FIH inactivated by slow autohydroxylation. Autohydroxylated FIH could not be reactivated by dithiothreitol (DTT) nor ascorbate, suggesting that autohydroxylation is likely to be irreversible under physiological conditions.

The higher order structure (HOS) of proteins plays a critical role in the efficacy and stability of biological drugs. Perturbation of the regional structure of proteins can affect biological activity and cause instability. Characterization of HOS has become an integral part of biological drug development and is expected from regulatory agencies. The commonly used techniques for HOS characterization, such as circular dichroism, Fourier-transform infrared, differential scanning calorimetry, intrinsic fluorescence, and hydrogen−deuterium exchange mass spectrometry, have their limitations ranging from lack of sensitivity and specificity to the need of high-level expertise and poor access to instrumentation due to high cost. In this study, we demonstrated a novel controlled proteolysis-based LC-QDa method for the detection of HOS change. By digesting proteins directly without denaturation and reduction, the HOS information can be revealed through the digested peptides. After optimizing the digestion conditions and the detection procedures, we identified 13 signature peptides that can monitor various antibody domains for any HOS changes caused by external stress. By comparing the peptide peak areas between unknown samples and a native control sample, any regional structural changes in unknown samples can be detected. The method was subsequently applied to a wide range of forced degradation samples to demonstrate higher sensitivity compared to the near-UV CD method that is frequently used for monitoring tertiary structural changes. By further reducing the number of signature peptides to five and optimizing liquid chromatography gradient duration, a streamlined, high-throughput, and controlled proteolysis method was successfully established. This method can be used to support process and formulation development as well as potentially for stability testing.

The clinical benefits of treatments with a combination of two or more therapeutic monoclonal antibodies (mAbs) have emerged in recent years. Imaged capillary isoelectric focusing is a frequently used technology in the biopharmaceutical industry for charge variant analysis of protein therapeutics. However, with the wide concentration ranges of combination products, one component may fall within the linear detection range, whereas the other does not. Here, we report a novel methodology to explore charge variants of mAb mixtures using multiple detection techniques simultaneously. We use ultraviolet absorbance to monitor the charge variants of the high-concentration component and native fluorescence (FL) to monitor the variants of the low-concentration one. Charge variants of mixtures that span 40-fold in ratio differences can be accurately quantified with this approach. In contrast to the conventional methods, it is not necessary to prepare and analyze two samples at different concentrations and combine the results for combination product testing. Additionally, the use of FL detection enables the charge variant analysis of highly potent/low abundant mAbs in a mixture. This methodology is more quality-control friendly and efficient for the charge variant analysis of combination products with wide ratios.