Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.

Insights into Nitrosoalkane Binding to Myoglobin Provided by Crystallography of Wild-Type and Distal Pocket Mutant Derivatives.

Nitrosoalkanes (R-N═O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH2) drugs or from the reduction of nitroorganics (RNO2). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe-RNO moieties remains limited. We report the preparation of ferrous wild-type and H64A sw MbII-RNO derivatives (λmax 424 nm; R = Me, Et, Pr, iPr) from the reactions of MbIII-H2O with dithionite and nitroalkanes. The apparent extent of formation of the wt Mb derivatives followed the order MeNO > EtNO > PrNO > iPrNO, whereas the order was the opposite for the H64A derivatives. Ferricyanide oxidation of the MbII-RNO derivatives resulted in the formation of the ferric MbIII-H2O precursors with loss of the RNO ligands. X-ray crystal structures of the wt MbII-RNO derivatives at 1.76-2.0 Å resoln. revealed N-binding of RNO to Fe and the presence of H-bonding interactions between the nitroso O-atoms and distal pocket His64. The nitroso O-atoms pointed in the general direction of the protein exterior, and the hydrophobic R groups pointed toward the protein interior. X-ray crystal structures for the H64A mutant derivatives were determined at 1.74-1.80 Å resoln. An analysis of the distal pocket amino acid surface landscape provided an explanation for the differences in ligand orientations adopted by the EtNO and PrNO ligands in their wt and H64A structures. Our results provide a good baseline for the structural analysis of RNO binding to heme proteins possessing small distal pockets.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Water Networks and Correlated Motions in Mutant Isocitrate Dehydrogenase 1 (IDH1) Are Critical for Allosteric Inhibitor Binding and Activity.

Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP+-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP+ binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.

A Tie2 kinase mutation causing venous malformations increases phosphorylation rates and enhances cooperativity.

The endothelial receptor tyrosine kinase Tie2 plays an important role in vascular formation and maintenance. Mutations in Tie2 lead to vascular malformations, which are painful vascular lesions that cause disfigurement, bleeding, and thrombosis. R849W Tie2 is the most common mutation implicated in an inherited form of vascular malformations and has been shown to be activating, though little is known about the kinetic features of catalysis. Here we undertake a steady-state kinetic analysis of heterologously expressed and purified wild type (WT) and R849W Tie2. While the catalytic efficiencies of the two forms are not significantly different, the observed maximal rate of phosphorylation, kcat,obs, is > 3-fold higher for R849W Tie2 compared to WT. Notably, steady-state catalysis by R849W Tie2 has more striking sigmoidal features compared to WT, suggesting enhanced positive cooperativity. We propose that activating catalytic features are one important consequence of the R849W mutation, though likely other factors such as increased protein binding affinity also contribute to the phenotypes observed in patients.

Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient's therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.

Molecular mechanisms of isocitrate dehydrogenase 1 (IDH1) mutations identified in tumors: The role of size and hydrophobicity at residue 132 on catalytic efficiency.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate (ICT) to α-ketoglutarate (αKG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of αKG to d-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit αKG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ∼640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas.

Development and Validation of a Phenotypic High-Content Imaging Assay for Assessing the Antiviral Activity of Small-Molecule Inhibitors Targeting Zika Virus.

Zika virus (ZIKV) has been linked to the development of microcephaly in newborns, as well as Guillain-Barré syndrome. There are currently no drugs available to treat ZIKV infection, and accordingly, there is an unmet medical need for the discovery of new therapies. High-throughput drug screening efforts focusing on indirect readouts of cell viability are prone to a higher frequency of false positives in cases where the virus is viable in the cell but the cytopathic effect (CPE) is reduced or delayed. Here, we describe a fast and label-free phenotypic high-content imaging assay to detect cells affected by the virus-induced CPE using automated imaging and analysis. Protection from the CPE correlates with a decrease in viral antigen production, as observed by immunofluorescence. We trained our assay using a collection of nucleoside analogues with activity against ZIKV; the previously reported antiviral activities of 2'-C-methylribonucleosides and ribavirin against the Zika virus in Vero cells were confirmed using our developed method. To validate the ability of our assay to reveal new anti-ZIKV compounds, we profiled a novel library of 24 natural product derivatives and found compound 1 to be an inhibitor of the ZIKV-induced cytopathic effect; the activity of the compound was confirmed in human fetal neural stem cells (NSCs). The described technique can be easily leveraged as a primary screening assay for profiling of the activities of large compound libraries against ZIKV and can be expanded to other ZIKV strains and other cell lines displaying morphological changes upon ZIKV infection.

Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ.

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3'-dideoxy-5-fluoro-3'-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3'-dideoxy-3'-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

Fluorescence resonance energy transfer studies of DNA polymerase ß: the critical role of fingers domain movements and a novel non-covalent step during nucleotide selection.

During DNA repair, DNA polymerase ß (Pol ß) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol ß fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol ß. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.

Mutations in human DNA polymerase γ confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients.

Human mitochondrial DNA polymerase γ (pol γ) is solely responsible for the replication and repair of the mitochondrial genome. Unsurprisingly, alterations in pol γ activity have been associated with mitochondrial diseases such as Alpers syndrome and progressive external ophthalmoplegia. Thus far, predicting the severity of mitochondrial disease based the magnitude of deficiency in pol γ activity has been difficult. In order to understand the relationship between disease severity in patients and enzymatic defects in vitro, we characterized the molecular mechanisms of four pol γ mutations, A957P, A957S, R1096C and R1096H, which have been found in patients suffering from aggressive Alpers syndrome to mild progressive external ophthalmoplegia. The A957P mutant showed the most striking deficiencies in the incorporation efficiency of a correct deoxyribonucleotide triphosphate (dNTP) relative to wild-type pol γ, with less, but still significant incorporation efficiency defects seen in R1096H and R1096C, and only a small decrease in incorporation efficiency observed for A957S. Importantly, this trend matches the disease severity observed in patients very well (approximated as A957P ≫ R1096C ≥ R1096H ≫ A957S, from most severe disease to least severe). Further, the A957P mutation conferred a two orders of magnitude loss of fidelity relative to wild-type pol γ, indicating that a buildup of mitochondrial genomic mutations may contribute to the death in infancy seen with these patients. We conclude that characterizing the unique molecular mechanisms of pol γ deficiency for physiologically important mutant enzymes is important for understanding mitochondrial disease and for predicting disease severity.

Catalytically distinct IDH1 mutants tune phenotype severity in tumor models.

Mutations in isocitrate dehydrogenase 1 (IDH1) impart a neomorphic reaction that produces the oncometabolite D-2-hydroxyglutarate (D2HG), which can inhibit DNA and histone demethylases to drive tumorigenesis via epigenetic changes. Though heterozygous point mutations in patients primarily affect residue R132, there are myriad D2HG-producing mutants that display unique catalytic efficiency of D2HG production. Here, we show that catalytic efficiency of D2HG production is greater in IDH1 R132Q than R132H mutants, and expression of IDH1 R132Q in cellular and mouse xenograft models leads to higher D2HG concentrations in cells, tumors, and sera compared to R132H-expressing models. Reduced representation bisulfite sequencing (RRBS) analysis of xenograft tumors shows expression of IDH1 R132Q relative to R132H leads to hypermethylation patterns in pathways associated with DNA damage. Transcriptome analysis indicates that the IDH1 R132Q mutation has a more aggressive pro-tumor phenotype, with members of EGFR, Wnt, and PI3K signaling pathways differentially expressed, perhaps through non-epigenetic routes. Together, these data suggest that the catalytic efficiency of IDH1 mutants modulate D2HG levels in cellular and in vivo models, resulting in unique epigenetic and transcriptomic consequences where higher D2HG levels appear to be associated with more aggressive tumors.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Oxidation of dihydrotestosterone by human cytochromes P450 19A1 and 3A4.

Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy- and 19-oxo derivatives and the resulting Δ(1,10)-, Δ(5,10)-, and Δ(9,10)-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ~10-fold higher than reported for 3α-reduction by 3α-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5ß-, and 9ß-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the Δ(1,10)-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the Δ(4,5) bond is remarkable in shifting the course of hydroxylation from the 6ß-, 2ß-, 1ß-, and 15ß-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure.

Balancing antiviral potency and host toxicity: identifying a nucleotide inhibitor with an optimal kinetic phenotype for HIV-1 reverse transcriptase.

Two novel thymidine analogs, 3'-fluoro-3'-deoxythymidine (FLT) and 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre-steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol γ, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2',3'-didehydro-2',3'-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4'-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2',3'-didehydro-2',3'-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.

Mechanism of interaction of human mitochondrial DNA polymerase γ with the novel nucleoside reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine indicates a low potential for host toxicity.

The potent antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a promising experimental agent for treating HIV infection. Pre-steady-state kinetics were used to characterize the interaction of EFdA-triphosphate (EFdA-TP) with human mitochondrial DNA polymerase γ (Pol γ) to assess the potential for toxicity. Pol γ incorporated EFdA-TP 4,300-fold less efficiently than dATP, with an excision rate similar to ddATP. This strongly indicates EFdA is a poor Pol γ substrate, suggesting minimal Pol γ-mediated toxicity, although this should be examined under clinical settings.

Probing altered enzyme activity in the biochemical characterization of cancer.

Enzymes have evolved to catalyze their precise reactions at the necessary rates, locations, and time to facilitate our development, to respond to a variety of insults and challenges, and to maintain a healthy, balanced state. Enzymes achieve this extraordinary feat through their unique kinetic parameters, myriad regulatory strategies, and their sensitivity to their surroundings, including substrate concentration and pH. The Cancer Genome Atlas (TCGA) highlights the extraordinary number of ways in which the finely tuned activities of enzymes can be disrupted, contributing to cancer development and progression often due to somatic and/or inherited genetic alterations. Rather than being limited to the domain of enzymologists, kinetic constants such as kcat, Km, and kcat/Km are highly informative parameters that can impact a cancer patient in tangible ways-these parameters can be used to sort tumor driver mutations from passenger mutations, to establish the pathways that cancer cells rely on to drive patients' tumors, to evaluate the selectivity and efficacy of anti-cancer drugs, to identify mechanisms of resistance to treatment, and more. In this review, we will discuss how changes in enzyme activity, primarily through somatic mutation, can lead to altered kinetic parameters, new activities, or changes in conformation and oligomerization. We will also address how changes in the tumor microenvironment can affect enzymatic activity, and briefly describe how enzymology, when combined with additional powerful tools, and can provide us with tremendous insight into the chemical and molecular mechanisms of cancer.