Substrate Specificity of Human Glycinamide Ribonucleotide Transformylase

Antle, Vincent D.; Donat, N.; Mei, Hua; Liao, Pei Ling; Vince, Robert; Caperelli, Carol A.

doi:10.1006/abbi.1999.1428

Cited by 3 publications

(3 citation statements)

References 22 publications

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“…10-formyl-5,8-dideazafolate (fDDF) and 10-acetyl-5,8-dideazafolate (aDDF) were prepared and quantitated as described previously (41,42). GAR was prepared by the method of Boschelli et al (43), purified by anion exchange chromatography (40), and the concentration of the β-anomer was determined by enzymatic assay with excess fDDF. Recombinant His-tagged TEV protease was produced and purified as described (44).…”

Section: Methodsmentioning

confidence: 99%

“…To date, few mechanistic or structural studies have been reported for human GART. These include the structures of the apo and ternary complex of rhGART (28), the structures of rhGART at low (pH 4.2) and high (pH 8.5) pH and in a binary complex with substrate β-GAR (27), nucleotide substrate specificity studies (40), structure-based inhibitor design and evaluation (29)(30)(31)(32)(33)(34)(35)(36)(37), and limited site-directed mutagenesis (38) of two of the wholly conserved residues (H108 and D144) implicated in the catalytic mechanism of the E. coli and human enzyme. The most recent inhibitor studies reinforce the conclusion that there are significant differences between E. coli and human GART.…”

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confidence: 99%

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Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Manieri¹,

Moore²,

Soellner³

et al. 2007

Biochemistry

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Human glycinamide ribonucleotide transformylase (GART) (EC2.1.2.2) 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, D144, and the semi-conserved 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 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 E. coli GART, previously used as a model for the human enzyme, were evident.Glycinamide ribonucleotide transformylase (GART 1 , EC 2.1.2.2) catalyzes the transfer of the formyl group from N10-formyl tetrahydrofolate to the primary, side-chain amino group of glycinamide ribonucleotide (GAR) to yield formyl-glycinamide ribonucleotide (FGAR) and tetrahydrofolate (Scheme 1), ultimately resulting in the incorporation of C-8 into inosinic acid (IMP). This reaction is the third step and the first of two folate-dependent formyl transfers in the de novo purine biosynthetic pathway. GART was first discovered and partially characterized from pigeon liver in pioneering investigations by Warren and Buchanan (1).The critical role that purine nucleotides play as precursors to RNA and DNA led to the suggestion that inhibition of de novo purine biosynthesis might be a viable approach toward cancer chemotherapy (2-4). This suggestion was confirmed when it was demonstrated that 5,10-dideazatetrahydrofolate, a potent anti-tumor agent, has, as its mechanism of action, the inhibition of GART and, consequently, of de novo purine biosynthesis (5).This discovery led to a resurgence of interest in the de novo purine biosynthetic pathway and served as the impetus for numerous studies on the mechanism (6-9), structure (10-15), and † Supported by NIH grant GM61194. 1 ABBREVIATIONS: GART, glycinamide ribonucleotide transformylase; GAR, glycinamide ribonucleotide; FGAR, formylglycinamide ribonucleotide; GARS, glycinamide ribonucleotide synthetase; AIRS, aminoimidazole ribonucleotide synthetase; rhGART, recombinant human glycinamide ribonucleotide transformylase; fDDF, 10-formyl-5,8-dideazafolate; aDDF, 10-acetyl-5,8-dideazafolate; GAR-OH, hydroxyacetamide ribonucleotide. 23,24). In contrast, human GART comprises the C-terminal domain of a large (108 kDa), trifunctional enzyme that also catalyzes the synthesis of GAR (GARS) and the synthesis of aminoimidazole ribonucleotide (AIRS) (25,26). These additional activities catalyze steps 2 and 5 of the pathway. NIH Public AccessThe E. coli studies have provided useful information, including the identification of the wholly conserved residues, N106, H108...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Manieri¹,

Moore²,

Soellner³

et al. 2007

Biochemistry

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“…Carbocyclic ribofuranosylamine ( 1 ), − in which a cyclopentane replaces the ribofuranose ring of the parent sugar, is a synthetic precursor for biologically active carbocyclic nucleosides, such as aristeromycin, − and for carbocyclic analogues of phosphosugar intermediates in purine biosynthesis. , It is also related to the synthetic precursors of the anti-retroviral drugs carbovir and abacavir …”

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Carbocyclic Ribosylamines: Synthesis of 5-Substituted Carbocyclic β-Ribofuranosylamines

2006

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A synthesis of 5-substituted cyclopentylamine precursors for 5'-substituted carbocyclic nucleoside analogues was developed. We show that the stereochemistry of the OsO4-catalyzed hydroxylation of an apically brominated lactam, 7-bromo-2-azabicyclo[2.2.1]hept-5-en-3-one, can be controlled through the appropriate selection of the lactam N-H protecting group. Sterically large groups direct the hydroxylation to the exo-face of the olefin, yielding hydroxylation products that can be converted into analogues of carbocyclic ribosides. Conversely, a sterically small protecting group permits OsO4 approach from the endo-face, yielding hydroxylation products analogous to carbocyclic lyxosides. A key intermediate for carbocyclic sugar production, (1S,2S,3R, 4R,5S)-1-(tert-butyloxycarbonyl)amino-5-bromo-2,3-(dimethylmethylene)dioxy-4-hydroxymethylcyclopentane, was synthesized starting from a commercially available enantiomerically pure lactam, (1S)-(+)-2-azabicyclo[2.2.1]hept-5-en-3-one, in seven steps in an overall yield of 21%.

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Phosphoribosylglydnamide formyltransferase

Springer Handbook of Enzymes

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Substrate Specificity of Human Glycinamide Ribonucleotide Transformylase

Cited by 3 publications

References 22 publications

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Carbocyclic Ribosylamines: Synthesis of 5-Substituted Carbocyclic β-Ribofuranosylamines

Phosphoribosylglydnamide formyltransferase

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