Evaluation of the Kinetic Mechanism of <i>Escherichia coli</i> Glycinamide Ribonucleotide Transformylase

Shim, Jae Hoon; Benkovic, Stephen J.

doi:10.1021/bi980244k

Cited by 30 publications

(32 citation statements)

References 22 publications

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“…In contrast, our apo (unoccupied GAR site) and ternary complex structures at pH 6.3 (enzyme active) showed essentially identical, ordered structures for this loop. Our binding studies with WT enzyme conducted at pH 8, the pH for maximum activity, showed independent binding for GAR and fDDF, suggesting that folate binding is not a prerequisite for organizing the active site for productive substrate binding and catalysis, which is consistent with our (see above) and other kinetic studies (8,50) which yielded a random sequential kinetic mechanism.…”

Section: Discussionsupporting

confidence: 91%

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: Discussionsupporting

confidence: 91%

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Manieri¹,

Moore²,

Soellner³

et al. 2007

Biochemistry

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“…This strategy, which mimics evolutionary processes of domain swapping, should complement existing strategies of directed evolution (32,33). GAR and fDDF were obtained and kinetic measurements were performed as described (30). Wild-type E. coli PurN was prepared as described by using a pMSW2 vector in MW12 cells (19).…”

Section: Resultsmentioning

confidence: 99%

Combinatorial protein engineering by incremental truncation

Ostermeier

Nixon

Shim

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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125

104

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We have developed a combinatorial approach, using incremental truncation libraries of overlapping N-and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer. This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding. We have tested this methodology on Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation. Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor. Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected.

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“…Restriction enzymes were purchased from either New England Biolabs or Promega; T4 DNA ligase, Calf Intestinal Alkaline Phosphatase, DNA polymerase I Klenow fragment, and Polynucleotide Kinase were from New England Biolabs. N 10 -formyl-5,8-dideazafolate (fDDF) was bought from the John Hynes Laboratory and GAR was synthesized as described (5). G-75 resin was from Sigma, Q-Sepharose resin from Amersham Pharmacia, and nickel-nitrilotriacetic acid from Qiagen (Chatsworth, CA).…”

Section: Methodsmentioning

confidence: 99%

“…These two enzymes carry out similar chemistry in catalyzing the transfer of a formyl group from 10-formyltetrahydrofolate to the amino group of the substrates GAR and AICAR to form fGAR and fAICAR. Investigators have invested their efforts in understanding the mechanisms of the two transformylases with the express purpose of rationally designing inhibitors that specifically target them, and in so doing disrupt purine biosynthesis (4)(5)(6)(7)(8). These and other purine enzymes may function in concert as a multienzyme complex, thus providing another target for chemotherapeutic intervention.…”

mentioning

confidence: 99%

Localization of GAR transformylase in Escherichia coli and mammalian cells

Gooljarsingh¹,

Ramcharan²,

Gilroy³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Enzymes of the de novo purine biosynthetic pathway may form a multienzyme complex to facilitate substrate flux through the ten serial steps constituting the pathway. One likely strategy for complex formation is the use of a structural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein-protein interactions. To ascertain whether this strategy pertains to the de novo purine enzymes, the localization pattern of the third purine enzyme, glycinamide ribonucleotide transformylase (GAR Tfase) was monitored in live Escherichia coli and mammalian cells. Genes encoding human as well as E. coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated expression of proteins and localization patterns monitored by using confocal fluorescence microscopy. In both instances images showed proteins to be diffused throughout the cytoplasm. Thus, GAR Tfase is not localized to an existing cellular architecture, so this device is probably not used to concentrate the members of the pathway. However, discrete clusters of the pathway may still exist throughout the cytoplasm.

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Evaluation of the Kinetic Mechanism of Escherichia coli Glycinamide Ribonucleotide Transformylase

Cited by 30 publications

References 22 publications

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Combinatorial protein engineering by incremental truncation

Localization of GAR transformylase in Escherichia coli and mammalian cells

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