Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Building on a theme: The redox hierarchy of pyridine nucleotide-disulfide oxidoreductases.

Flavin disulfide reductases (FDRs) are FAD-dependent enzymes that transmit electrons from NAD(P)H to reduce specific oxidant substrate disulfides. These enzymes have been studied extensively, most particularly the paradigm examples: glutathione reductase and thioredoxin reductase. The common, though not universal, traits of the family include a tyrosine- or phenylalanine-gated binding pocket for NAD(P) nicotinamides adjacent to the FAD isoalloxazine re-face, and a disulfide stacked against the si-face of the isoalloxazine whose dithiol form is activated for subsequent exchange reactions by a nearby histidine acting as a base. This arrangement promotes transduction of the reducing equivalents for disulfide exchange relay reactions. From an observational standpoint the proximal parallel stacking of three redox moieties induces up to three opportunities for unique charge transfer interactions (NAD(P)H FAD, NAD(P)+•FADH2, and FAD•thiolate). In transient state, the charge transfer transitions provide discrete signals to assign reaction sequences. This review summarizes the lineage of observations for the FDR enzymes that have been extensively studied. Where applicable and in order to chart a consistent interpretation of the record, only data derived from studies that used anaerobic methods are cited. These data reveal a recurring theme for catalysis that is elaborated with specific additional functionalities for each oxidant substrate.

Assigning function to active site residues of Schistosoma mansoni thioredoxin/glutathione reductase from analysis of transient state reductive half-reactions with variant forms of the enzyme.

Thioredoxin/glutathione reductase (TGR) from the platyhelminthic parasitic worms has recently been identified as a drug target for the treatment of schistosomiasis. Schistosomes lack catalase, and so are heavily reliant on the regeneration of reduced thioredoxin (Trx) and glutathione (GSH) to reduce peroxiredoxins that ameliorate oxidative damage from hydrogen peroxide generated by the host immune response. This study focuses on the characterization of the catalytic mechanism of Schistosoma mansoni TGR (SmTGR). Variant forms of SmTGR were studied to assign the function of residues that participate in the electron distribution chain within the enzyme. Using anaerobic transient state spectrophotometric methods, redox changes for the FAD and NADPH were observed and the function of specific residues was defined from observation of charge transfer absorption transitions that are indicative of specific complexations and redox states. The C159S variant prevented distribution of electrons beyond the flavin and as such did not accumulate thiolate-FAD charge transfer absorption. The lack of this absorption facilitated observation of a new charge transfer absorption consistent with proximity of NADPH and FAD. The C159S variant was used to confine electrons from NADPH at the flavin, and it was shown that NADPH and FAD exchange hydride in both directions and come to an equilibrium that yields only fractional FAD reduction, suggesting that both have similar reduction potentials. Mutation of U597 to serine resulted in sustained thiolate-FAD charge transfer absorption and loss of the ability to reduce Trx, indicating that the C596-U597 disulfide functions in the catalytic sequence to receive electrons from the C154 C159 pair and distribute them to Trx. No kinetic evidence for a loss or change in function associated with the distal C28-C31 disulfide was observed when the C31S variant reductive half-reaction was observed. The Y296A variant was shown to slow the rate of but increase extent of reduction of the flavin, and the dissociation of NADP+. The H571 residue was confirmed to be the residue responsible for the deprotonation of the C159 thiol, increasing its reactivity and generating the prominent thiolate-FAD charge transfer absorption that accumulates with oxidation of the flavin.

Dihydropyrmidine dehydrogenase from Escherichia coli: Transient state analysis reveals both reductive activation prior to turnover and diminished substrate effector roles relative to the mammalian form.

Dihydropyrimidine dehydrogenase (DPD) is an enzyme that uses an elaborate architecture to catalyze a simple net reaction: the reduction of the vinylic bond of uracil and thymine. Known DPDs have two active sites separated by approximately 60 Å. One active site has an FAD cofactor and binds NAD(P) and the other has an FMN cofactor and binds pyrimidines. The intervening distance is spanned by four Fe4S4 centers that act as an electron conduit. Recent advancements with porcine DPD have revealed unexpected chemical sequences where the enzyme undergoes reductive activation by transferring two electrons from NADPH to the FMN via the FAD such that the active form has the cofactor set FAD•4(Fe4S4)•FMNH2. Here we describe the first comprehensive kinetic investigation of a bacterial form of DPD. Using primarily transient state methods, DPD from E. coli (EcDPD) was shown to have a similar mechanism to that observed with the mammalian form in that EcDPD is observed to undergo reductive activation before pyrimidine reduction and displays half-of-sites activity. However, two distinct aspects of the EcDPD reaction relative to the mammalian enzyme were observed that relate to the effector roles for substrates: (i) the enzyme will rapidly take up electrons from NADH, reducing a flavin in the absence of pyrimidine substrate, and (ii) the activated form of the enzyme can become fully oxidized by transferring electrons to pyrimidine substrates in the absence of NADH.

The unusual chemical sequences of mammalian dihydropyrimidine dehydrogenase revealed by transient-state analysis.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of the 5,6-vinylic bond of uracil and thymine with electrons from NADPH. The complexity of the enzyme belies the simplicity of the reaction catalyzed. To accomplish this chemistry DPD has two active sites that are ∼60Šapart, both of which house flavin cofactors, FAD and FMN. The FAD site interacts with NADPH, while the FMN site with pyrimidines. The distance between the flavins is spanned by four Fe4S4 centers. Though DPD has been studied for nearly 50years, it is only recently that the novel apects of its mechanism have been described. The primary reason for this is that the chemistry of DPD is not portrayed adequately by known descriptive steady-state mechanism categories. The highly chromophoric nature of the enzyme has recently been exploited in transient-state to document unexpected reaction sequences. Specifically, DPD undergoes reductive activation prior to catalytic turnover. Two electrons are taken up from NADPH and transmitted via the FAD and Fe4S4 centers to form the FAD•4(Fe4S4)•FMNH2 form of the enzyme. This form of the enzyme will only reduce pyrimidine substrates in the presence NADPH, establishing that hydride transfer to the pyrimidine precedes reductive reactivation that reinstates the active form of the enzyme. DPD is therefore the first flavoprotein dehydrogenase known to complete the oxidative half-reaction prior to the reductive half-reaction. Here we describe the methods and deduction that led to this mechanistic assignment.

Descriptive Analysis of Transient-State Observations for Thioredoxin/Glutathione Reductase (Sec597Cys) from Schistosoma mansoni.

Thioredoxin/glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both oxidized thioredoxin and glutathione with electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH). SmTGR is a drug target for the treatment of Schistosomiasis, an infection caused by Schistosoma platyhelminths residing in the blood vessels of the host. Schistosoma spp. are reliant on TGR enzymes as they lack catalase and so use reduced thioredoxin and glutathione to regenerate peroxiredoxins consumed in the detoxification of reactive oxygen species. SmTGR is a flavin adenine dinucleotide (FAD)-dependent enzyme, and we have used the flavin as a spectrophotometric reporter to observe the movement of electrons within the enzyme. The data show that NADPH fractionally reduces the active site flavin with an observed rate constant estimated in this study to be ∼3000 s-1. The flavin then reoxidizes by passing electrons at a similar rate to the proximal Cys159-Cys154 disulfide pair. The dissociation of NADP+ occurs with a rate of ∼180 s-1, which induces the deprotonation of Cys159, and this coincides with the accumulation of an intense FAD-thiolate charge transfer band. It is proposed that the electrons then pass to the Cys596-Cys597 disulfide pair of the associated subunit in the dimer with a net rate constant of ∼2 s-1. (Note: Cys597 is Sec597 in wild-type (WT) SmTGR.) From this position, the electrons can be passed to oxidized thioredoxin or further into the protein to reduce the Cys28-Cys31 disulfide pair of the originating subunit of the dimer. From the Cys28-Cys31 center, electrons can then pass to oxidized glutathione that has a binding site directly adjacent.

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid.

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.

Mammalian dihydropyrimidine dehydrogenase: Added mechanistic details from transient-state analysis of charge transfer complexes.

Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by ∼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four Fe4S4 centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the Fe4S4 centers to yield the active state with a cofactor set of FAD•4(Fe4S4)•FMNH2. The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP+ indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzyme•pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP+ DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.

Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis.

RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excises the C4 of d-ribulose-5-phosphate (d-Ru5P) as formate. RibB generates the four-carbon substrate for lumazine synthase that is incorporated into the xylene moiety of lumazine and ultimately the riboflavin isoalloxazine. The reaction was first identified by Bacher and co-workers in the 1990s, and their chemical mechanism hypothesis became canonical despite minimal direct evidence. X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues, and the consensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentrations equal to the enzyme concentration. This was confirmed by electron paramagnetic resonance of the enzyme reconstituted with manganese and crystal structures liganded with Mn2+ and a variety of sugar phosphates. Two transient species prior to the formation of products were identified using acid quench of single turnover reactions in combination with NMR for singly and fully 13C-labeled d-Ru5P. These data indicate that dehydration of C1 forms the first transient species, which undergoes rearrangement by a 1,2 migration, fusing C5 to C3 and generating a hydrated C4 that is poised for elimination as formate. Structures determined from time-dependent Mn2+ soaks of VcRibB-d-Ru5P crystals show accumulation in crystallo of the same intermediates. Collectively, these data reveal for the first time crucial transient chemical states in the mechanism of RibB.

Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.

Rational Design, Synthesis, and Mechanism of (3S,4R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.

Mammalian dihydropyrimidine dehydrogenase.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the two-electron reduction of pyrimidine bases uracil and thymine as the first step in pyrimidine catabolism. The enzyme achieves this simple chemistry using a complex cofactor set including two flavins and four Fe4S4 centers. The flavins, FAD and FMN, interact with respective NADPH and pyrimidine substrates and the iron-sulfur centers form an electron transfer wire that links the two active sites that are separated by 56 Å. DPD accepts the common antineoplastic agent 5-fluorouracil as a substrate and so undermines the establishment of efficacious toxicity. Though studied for multiple decades, a precise description of the behavior of the enzyme had remained elusive. It was recently shown that the active form of DPD has the cofactor set of FAD-4(Fe4S4)-FMNH2. This two-electron reduced state is consistent with fewer mechanistic possibilities and data suggests that the instigating and rate determining step in the catalytic cycle is reduction of the pyrimidine substrate that is followed by relatively rapid oxidation of NADPH at the FAD that, via the electron conduit of the 4(Fe4S4) centers, reinstates the FMNH2 cofactor for subsequent catalytic turnover.

Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of E. coli GTP Cyclohydrolase II.

Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8-N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α-ß phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-ß-pyrimidinone 5'-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity in Escherichia coli (Foor, F., Brown, G. M. J. Biol. Chem., 1975, 250, 9, 3545-3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA from E. coli with the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point 31P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation.

Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase.

The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.

Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase.

The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 Šthat are bridged by four Fe4S4 centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020) Biochemistry 59, 2419-2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4 centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.

The Interaction of Porcine Dihydropyrimidine Dehydrogenase with the Chemotherapy Sensitizer: 5-Ethynyluracil.

Dihydropyrimidine dehydrogenase (DPD) is a complex enzyme that reduces the 5,6-vinylic bond of pyrimidines, uracil, and thymine. 5-Fluorouracil (5FU) is also a substrate for DPD and a common chemotherapeutic agent used to treat numerous cancers. The reduction of 5FU to 5-fluoro-5,6-dihydrouracil negates its toxicity and efficacy. Patients with high DPD activity levels typically have poor outcomes when treated with 5FU. DPD is thus a central mitigating factor in the treatment of a variety of cancers. 5-Ethynyluracil (5EU) covalently inactivates DPD by cross-linking with the active-site general acid cysteine in the pyrimidine binding site. This reaction is dependent on the simultaneous binding of 5EU and nicotinamide adenine dinucleotide phosphate (NADPH). This ternary complex induces DPD to become activated by taking up two electrons from the NADPH. The covalent inactivation of DPD by 5EU occurs concomitantly with this reductive activation with a rate constant of ∼0.2 s-1. This kinact value is correlated with the rate of reduction of one of the two flavin cofactors and the localization of a mobile loop in the pyrimidine active site that places the cysteine that serves as the general acid in catalysis proximal to the 5EU ethynyl group. Efficient cross-linking is reliant on enzyme activation, but this process appears to also have a conformational aspect in that nonreductive NADPH analogues can also induce a partial inactivation. Cross-linking then renders DPD inactive by severing the proton-coupled electron transfer mechanism that transmits electrons 56 Šacross the protein.

Structural and Kinetic Analyses Reveal the Dual Inhibition Modes of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic Acid (BCF3).

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and the leading cause of death among people with cirrhosis. HCC is typically diagnosed in advanced stages when tumors are resistant to both radio- and chemotherapy. Human ornithine aminotransferase (hOAT) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme involved in glutamine and proline metabolism. Because hOAT is overexpressed in HCC cells and a contributing factor for the uncontrolled cellular division that propagates malignant tumors (Ueno et al. J. Hepatol. 2014, 61, 1080-1087), it is a potential drug target for the treatment of HCC. (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)-cyclopentane-1-carboxylic acid (BCF3) has been shown in animal models to slow the progression of HCC by acting as a selective and potent mechanism-based inactivator of OAT (Zigmond et al. ACS Med. Chem. Lett. 2015, 6, 840-844). Previous studies have shown that the BCF3-hOAT reaction has a bifurcation in which only 8% of the inhibitor inactivates the enzyme while the remaining 92% ultimately acts as a substrate and undergoes hydrolysis to regenerate the active PLP form of the enzyme. In this manuscript, the rate-limiting step of the inactivation mechanism was determined by stopped-flow spectrophotometry and time-dependent 19F NMR experiments to be the decay of a long-lived external aldimine species. A crystal structure of this transient complex revealed both the structural basis for fractional irreversible inhibition and the principal mode of inhibition of hOAT by BCF3, which is to trap the enzyme in this transient but quasi-stable external aldimine form.