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Stephen J. Benkovic - One of the best experts on this subject based on the ideXlab platform.

  • On the structural and functional modularity of Glycinamide Ribonucleotide formyltransferases
    Protein science : a publication of the Protein Society, 2009
    Co-Authors: Seung-goo Lee, Stefan Lutz, Stephen J. Benkovic
    Abstract:

    Glycinamide Ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the Glycinamide Ribonucleotide substrate. Despite the low amino acid-sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable. As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids. The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain. From a pool of several dozen sequence distinct hybrids, six in vivo-functional fusion genes were selected, overexpressed, and purified to homogeneity. The kinetic analysis of these constructs and the comparison of their kcat and KM values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are “modular encoded” and can be exchanged by domain swapping. The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization.

  • purt encoded Glycinamide Ribonucleotide transformylase accommodation of adenosine nucleotide analogs within the active site
    Journal of Biological Chemistry, 2002
    Co-Authors: James B Thoden, Stephen J. Benkovic, Steven M Firestine, Hazel M Holden
    Abstract:

    Abstract PurT-encoded Glycinamide Ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of Glycinamide Ribonucleotide through a catalytic mechanism requiring Mg2+ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase fromEscherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg2+ATP, Mg2+-5′-adenylylimidodiphosphate, Mg2+-β,γ-methyleneadenosine 5′-triphosphate, Mg2+ATPγS, or Mg2+ADP. Taken together, these studies indicate that the conformation of the so-called “T-loop,” delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the “P-loop”-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.

  • PurT-encoded Glycinamide Ribonucleotide Transformylase ACCOMMODATION OF ADENOSINE NUCLEOTIDE ANALOGS WITHIN THE ACTIVE SITE
    The Journal of biological chemistry, 2002
    Co-Authors: James B Thoden, Stephen J. Benkovic, Steven M Firestine, Hazel M Holden
    Abstract:

    PurT-encoded Glycinamide Ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of Glycinamide Ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.

  • molecular structure of escherichia coli purt encoded Glycinamide Ribonucleotide transformylase
    Biochemistry, 2000
    Co-Authors: James B Thoden, Stephen J. Benkovic, Steven M Firestine, Andrew E Nixon, Hazel M Holden
    Abstract:

    In Escherichia coli, the PurT-encoded Glycinamide Ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of Glycinamide Ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli:  one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary...

  • Molecular structure of Escherichia coli PurT-encoded Glycinamide Ribonucleotide transformylase.
    Biochemistry, 2000
    Co-Authors: James B Thoden, Stephen J. Benkovic, Steven M Firestine, Andrew E Nixon, Hazel M Holden
    Abstract:

    In Escherichia coli, the PurT-encoded Glycinamide Ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of Glycinamide Ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in Ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMPPNP.

Ian A. Wilson - One of the best experts on this subject based on the ideXlab platform.

  • biological and structural evaluation of 10r and 10s methylthio ddacthf reveals a new role for sulfur in inhibition of Glycinamide Ribonucleotide transformylase
    Biochemistry, 2013
    Co-Authors: Stephen Connelly, Jessica K. Demartino, Dale L. Boger, Ian A. Wilson
    Abstract:

    Glycinamide Ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 A). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization revea...

  • Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of Glycinamide Ribonucleotide transformylase.
    Biochemistry, 2013
    Co-Authors: Stephen Connelly, Jessica K. Demartino, Dale L. Boger, Ian A. Wilson
    Abstract:

    Glycinamide Ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC₅₀ = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC₅₀ = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.

  • Biological and Structural Evaluation of 10R- and 10S‑Methylthio-DDACTHF Reveals a New Role for Sulfur in Inhibition of Glycinamide Ribonucleotide Transformylase
    2013
    Co-Authors: Stephen Connelly, Dale L. Boger, Jessica K. Demartino, Ian A. Wilson
    Abstract:

    Glycinamide Ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC50 = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC50 = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase

  • Asymmetric Synthesis of Inhibitors of Glycinamide Ribonucleotide Transformylase
    Journal of medicinal chemistry, 2008
    Co-Authors: Jessica K. Demartino, Ian A. Wilson, Inkyu Hwang, Stephen Connelly, Dale L. Boger
    Abstract:

    Glycinamide Ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway that require folate cofactors and has emerged as a productive target for antineoplastic therapeutic intervention. The asymmetric synthesis and evaluation of the two diastereomers of 10-methylthio-DDACTHF (10R-3 and 10S-3) and related analogues as potential inhibitors of GAR Tfase are reported. This work, which defines the importance of the C10 stereochemistry for this class of inhibitors of GAR Tfase, revealed that both diastereomers are potent inhibitors of rhGAR Tfase (10R-3 Ki = 210 nM, 10S-3 Ki = 180 nM) that exhibit effective cell growth inhibition (CCRF-CEM IC50 = 80 and 50 nM, respectively), which is dependent on intracellular polyglutamation by folylpolyglutamate synthetase (FPGS) but not intracellular transport by the reduced folate carrier.

  • Discovery of a potent, nonpolyglutamatable inhibitor of Glycinamide Ribonucleotide transformylase.
    Journal of medicinal chemistry, 2006
    Co-Authors: Jessica K. Demartino, Ian A. Wilson, Inkyu Hwang, Dale L. Boger
    Abstract:

    Glycinamide Ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway that require folate cofactors. Herein we report the discovery of a potent, nonpolyglutamatable, and selective inhibitor of GAR Tfase. Compound 12, which possesses a tetrazole in place of the gamma-carboxylic acid in the l-glutamate subunit of the potent GAR Tfase inhibitor 1, was active in cellular-based functional assays exhibiting purine-sensitive cytotoxic activity (IC(50) = 40 nM, CCRF-CEM) and was selective for inhibition of rhGAR Tfase (K(i) = 130 nM). Notably, 12 was only 2.5-fold less potent than 1 in cellular assays and 4-fold less potent against rhGAR Tfase. Like 1, this functional activity of 12 in the cell-based assay benefits from and requires transport into the cell by the reduced folate carrier but, unlike 1, is independent of folyl polyglutamate synthase (FPGS) expression levels and polyglutamation.

Carol A Caperelli - One of the best experts on this subject based on the ideXlab platform.

  • Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes
    Biochemistry, 2007
    Co-Authors: Wanda Manieri, Molly E. Moore, Matthew B. Soellner, Pearl Tsang, Carol A Caperelli
    Abstract:

    Human Glycinamide Ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.

  • The apo and ternary complex structures of a chemotherapeutic target: human Glycinamide Ribonucleotide transformylase.
    Biochemistry, 2005
    Co-Authors: Tanya E. S. Dahms, Eugene L. Giroux, Germaine Sainz, Carol A Caperelli, Janet L. Smith
    Abstract:

    : Glycinamide Ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylGlycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a Glycinamide Ribonucleotide analogue, hydroxyacetamide Ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.

  • Substrate specificity of human Glycinamide Ribonucleotide transformylase.
    Archives of biochemistry and biophysics, 1999
    Co-Authors: Vincent D. Antle, Nathaniel Donat, Mei Hua, Pei Ling Liao, Robert Vince, Carol A Caperelli
    Abstract:

    The nucleotide substrate specificity of human Glycinamide Ribonucleotide transformylase, a chemotherapeutic target, has been examined. The enzyme accepts the sarcosyl analog of Glycinamide Ribonucleotide, carbocyclic Glycinamide Ribonucleotide, and two phosphonate derivatives of carbocyclic Glycinamide Ribonucleotide with V/K values, relative to that obtained for beta-Glycinamide Ribonucleotide, of 1, 27, 1.4, and 2.9%, respectively. Several other analogs of carbocyclic Glycinamide Ribonucleotide, namely a truncated phosphonate and 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-carbocyclic Glycinamide Ribonucleotide, were inhibitors of the enzyme, competitive against Glycinamide Ribonucleotide, with Ki values approximately 100 times higher than the Km for -Glycinamide Ribonucleotide. Although the results of the present study parallel those obtained previously with the avian enzyme (V. D. Antle, D. Liu, B. R. McKellar, C. A. Caperelli, M. Hua, and R. Vince (1996) J. Biol. Chem. 271, 6045-6049), quantitative differences between the two enzyme species have been uncovered.

  • substrate specificity of human Glycinamide Ribonucleotide transformylase
    Archives of Biochemistry and Biophysics, 1999
    Co-Authors: Vincent D. Antle, Nathaniel Donat, Mei Hua, Pei Ling Liao, Robert Vince, Carol A Caperelli
    Abstract:

    Abstract Thenucleotide substrate specificity of human Glycinamide Ribonucleotide transformylase, a chemotherapeutic target, has been examined. The enzyme accepts the sarcosyl analog of Glycinamide Ribonucleotide, carbocyclic Glycinamide Ribonucleotide, and two phosphonate derivatives of carbocyclic Glycinamide Ribonucleotide with V / K values, relative to that obtained for β-Glycinamide Ribonucleotide, of 1, 27, 1.4, and 2.9%, respectively. Several other analogs of carbocyclic Glycinamide Ribonucleotide, namely a truncated phosphonate and 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-carbocyclic Glycinamide Ribonucleotide, were inhibitors of the enzyme, competitive against Glycinamide Ribonucleotide, with K i values approximately 100 times higher than the K m for β-Glycinamide Ribonucleotide. Although the results of the present study parallel those obtained previously with the avian enzyme (V. D. Antle, D. Liu, B. R. McKellar, C. A. Caperelli, M. Hua, and R. Vince (1996) J. Biol. Chem. 271, 6045–6049), quantitative differences between the two enzyme species have been uncovered.

  • The Human Trifunctional Enzyme ofde NovoPurine Biosynthesis: Heterologous Expression, Purification, and Preliminary Characterization
    Protein expression and purification, 1998
    Co-Authors: Mark T. Poch, Wen Qin, Carol A Caperelli
    Abstract:

    The cDNA for the human trifunctional enzyme of de novo purine biosynthesis, which encodes Glycinamide Ribonucleotide synthetase, aminoimidazole Ribonucleotide synthetase, and Glycinamide Ribonucleotide trans-formylase, has been overexpressed in Escherichia coli and its protein product has been purified to homogeneity. The Glycinamide Ribonucleotide transformylase activity, which constitutes the C-terminal domain of the trifunctional enzyme, has been characterized with respect to its kinetic constants, Vmax = 3.03 +/- 0.15 micromol/min-mg and Km values for beta-Glycinamide Ribonucleotide and 10-formyl-5,8-dideazafolate of 0.94 +/- 0.21 and 1.58 +/- 0.25 microM, respectively, and its kinetic mechanism, which is ordered-sequential with the folate substrate binding first. The correspondence of these data to those obtained for the Glycinamide Ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional.

Patricia A. Jennings - One of the best experts on this subject based on the ideXlab platform.

  • A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric Glycinamide Ribonucleotide transformylase at 1.8 to 1.9 A.
    Journal of molecular biology, 1998
    Co-Authors: Mason M. Yamashita, Jae Hoon Shim, Christine A Mullen, Patricia A. Jennings, Samantha Greasley, Steven J Benkovic, Ian A. Wilson
    Abstract:

    A mutation in the dimer interface of Escherichia coli Glycinamide Ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the pKa of His108 is in fact raised to 9. 2. The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.

  • Glycinamide Ribonucleotide Transformylase Undergoes pH-dependent Dimerization
    Journal of molecular biology, 1996
    Co-Authors: Christine A Mullen, Patricia A. Jennings
    Abstract:

    Glycinamide Ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5). We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state. In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering. The data give a best fit to a cooperative three-proton transfer mechanism: [formula: see text] A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization. Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization. Fitting of the normalized titration curves yields an apparent pKa = 7.16(+/-0.02) and a subnanomolar KD for the transition. Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization. There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization. Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution. Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell.

  • Glycinamide Ribonucleotide transformylase undergoes ph dependent dimerization
    Journal of Molecular Biology, 1996
    Co-Authors: Christine A Mullen, Patricia A. Jennings
    Abstract:

    Abstract Glycinamide Ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5). We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state. In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering. The data give a best fit to a cooperative three-proton transfer mechanism: 2M + 3H + ⇌MH 2 2+ + MH + ⇌DH 3 3+ A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization. Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization. Fitting of the normalized titration curves yields an apparent p K a =7.16(±0.02) and a subnanomolar K D for the transition. Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization. There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization. Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution. Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell.

Aleem Gangjee - One of the best experts on this subject based on the ideXlab platform.

  • Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase.
    Biochemistry, 2016
    Co-Authors: Siobhan M. Deis, Zhanjun Hou, Larry H Matherly, Aleem Gangjee, Arpit Doshi, Charles E. Dann
    Abstract:

    Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of Glycinamide Ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo[2,3-d]pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition.

  • Structural and Enzymatic Analysis of Tumor-Targeted Antifolates That Inhibit Glycinamide Ribonucleotide Formyltransferase
    2016
    Co-Authors: Siobhan M. Deis, Zhanjun Hou, Aleem Gangjee, Arpit Doshi, Larry H. Matherly, Charles E. Dann
    Abstract:

    Pemetrexed and methotrexate are antifolates used for cancer chemotherapy and inflammatory diseases. These agents have toxic side effects resulting, in part, from nonspecific cellular transport by the reduced folate carrier (RFC), a ubiquitously expressed facilitative transporter. We previously described 2-amino-4-oxo-6-substituted pyrrolo­[2,3-d]­pyrimidine antifolates with modifications of the side chain linker and aromatic ring that are poor substrates for RFC but are efficiently transported via folate receptors (FRs) and the proton-coupled folate transporter (PCFT). These targeted antifolates are cytotoxic in vitro toward FR- and PCFT-expressing tumor cells and in vivo with human tumor xenografts in immune-compromised mice, reflecting selective cellular uptake. Antitumor efficacy is due to inhibition of Glycinamide Ribonucleotide (GAR) formyltransferase (GARFTase) activity in de novo synthesis of purine nucleotides. This study used purified human GARFTase (formyltransferase domain) to assess in vitro inhibition by eight novel thieno- and pyrrolo­[2,3-d]­pyrimidine antifolates. Seven analogues (AGF23, AGF71, AGF94, AGF117, AGF118, AGF145, and AGF147) inhibited GARFTase with Ki values in the low- to mid-nanomolar concentration range, whereas AGF50 inhibited GARFTase with micromolar potency similar to that of PMX. On the basis of crystal structures of ternary complexes with GARFTase, β-GAR, and the monoglutamyl antifolates, differences in inhibitory potencies correlated well with antifolate binding and the positions of the terminal carboxylates. Our data provide a mechanistic basis for differences in inhibitory potencies between these novel antifolates and a framework for future structure-based drug design. These analogues could be more efficacious than clinically used antifolates, reflecting their selective cellular uptake by FRs and PCFT and potent GARFTase inhibition

  • Novel 5-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of Glycinamide Ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide Ribonucleotide formyltransferase and as potential antitumor agents.
    Journal of medicinal chemistry, 2015
    Co-Authors: Yiqiang Wang, Shermaine Mitchell-ryan, Zhanjun Hou, Larry H Matherly, Sudhir Raghavan, Christina George, Steven Orr, Aleem Gangjee
    Abstract:

    A new series of 5-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 6-11 with varying chain lengths (n = 1-6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo[2,3-d]pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at Glycinamide Ribonucleotide formyltransferase (GARFTase). Compounds 6-11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8-10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide Ribonucleotide formyltransferase. Our studies identify a unique structure-activity relationship for transport and dual target inhibition.

  • Novel 5‑Substituted Pyrrolo[2,3‑d]pyrimidines as Dual Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5‑Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase and as Potential Antitumor Agents
    2015
    Co-Authors: Yiqiang Wang, Shermaine Mitchell-ryan, Zhanjun Hou, Larry H Matherly, Sudhir Raghavan, Christina George, Steven Orr, Aleem Gangjee
    Abstract:

    A new series of 5-substituted thiopheneyl pyrrolo­[2,3-d]­pyrimidines 6–11 with varying chain lengths (n = 1–6) were designed and synthesized as hybrids of the clinically used anticancer drug pemetrexed (PMX) and our 6-substituted thiopheneyl pyrrolo­[2,3-d]­pyrimidines 2c and 2d with folate receptor (FR) α and proton-coupled folate transporter (PCFT) uptake specificity over the reduced folate carrier (RFC) and inhibition of de novo purine nucleotide biosynthesis at Glycinamide Ribonucleotide formyltransferase (GARFTase). Compounds 6–11 inhibited KB human tumor cells in the order 9 = 10 > 8 > 7 > 6 = 11. Compounds 8–10 were variously transported by FRα, PCFT, and RFC and, unlike PMX, inhibited de novo purine nucleotide rather than thymidylate biosynthesis. The antiproliferative effects of 8 and 9 appeared to be due to their dual inhibitions of both GARFTase and 5-aminoimidazole-4-carboxamide Ribonucleotide formyltransferase. Our studies identify a unique structure–activity relationship for transport and dual target inhibition

  • Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine antifolates as dual-acting inhibitors of Glycinamide Ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide Ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: i
    Journal of medicinal chemistry, 2013
    Co-Authors: Shermaine Mitchell-ryan, Christina Cherian, Yiqiang Wang, Zhanjun Hou, Larry H Matherly, Sudhir Raghavan, Steven Orr, Manasa P. Ravindra, Eric C. Hales, Aleem Gangjee
    Abstract:

    We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA Ribonucleotide formyltransferase (AICARFTase) and Glycinamide Ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.