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Stephen J. Benkovic - One of the best experts on this subject based on the ideXlab platform.
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Design, synthesis, and biological evaluation of simplified α-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase
Bioorganic & medicinal chemistry, 2003Co-Authors: Thomas H. Marsilje, Stephen J. Benkovic, Michael P. Hedrick, Joel Desharnais, Ali Tavassoli, Yan Zhang, Ian A. Wilson, Dale L. BogerAbstract:A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of Glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase).
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molecular structure of escherichia coli purt encoded Glycinamide ribonucleotide transformylase
Biochemistry, 2000Co-Authors: James B Thoden, Stephen J. Benkovic, Steven M Firestine, Andrew E Nixon, Hazel M HoldenAbstract: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...
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Design, synthesis, and biological evaluation of fluoronitrophenyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase.
Bioorganic & medicinal chemistry letters, 2000Co-Authors: Dale L. Boger, Jae Hoon Shim, Thomas H. Marsilje, Qing Jin, Michael P. Hedrick, Stephen J. Baker, René A. Castro, Stephen J. BenkovicAbstract:The examination results of a novel series of potential inhibitors of Glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. These agents incorporate an electrophilic fluoronitrophenyl group that can potentially react with an active site nucleophile or the substrate GAR/AICAR amine via nucleophilic aromatic substitution.
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Improvement in the efficiency of formyl transfer of a GAR transformylase hybrid enzyme.
Protein engineering, 2000Co-Authors: Andrew E Nixon, Stephen J. BenkovicAbstract:A hybrid Glycinamide ribonucleotide transformylase was assembled from two protein domains that were treated as discrete modules. One module contained the ribonucleotide binding domain from the purN Glycinamide ribonucleotide transformylase; the second module contained the catalytic machinery and the formyl tetrahydrofolate binding domain from the enzyme encoded by purU, formyl tetrahydrofolate hydrolase. The resultant enzyme showed 0.1% catalytic activity of the wild-type Glycinamide ribonucleotide transformylase enzyme but had a formyl transfer efficiency of 10%. A combinatorial mutagenesis approach was used to improve the solubility and formyl transfer properties of the hybrid enzyme. The mutagenized hybrid Glycinamide ribonucleotide transformylase was initially expressed as a fusion to the alpha-peptide of beta-galactosidase. Clones were selected for improvement in solubility by determining which clones were capable of alpha-complementation using a blue/white screen. One clone was further characterized and found to have an improved efficiency of transfer of the ribonucleotide increasing this property to >95%.
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Conformationally restricted analogues designed for selective inhibition of GAR Tfase versus thymidylate synthase or dihydrofolate reductase.
Bioorganic & medicinal chemistry, 2000Co-Authors: Dale L. Boger, Jae Hoon Shim, Marc A. Labroli, Thomas H. Marsilje, Qing Jin, Michael P. Hedrick, Stephen J. Baker, Stephen J. BenkovicAbstract:The synthesis and evaluation of a series of conformationally restricted analogues of 10-formyl-tetrahydrofolate as potential inhibitors of Glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide transformylase (AICAR Tfase) are reported.
Carol A Caperelli - One of the best experts on this subject based on the ideXlab platform.
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Substrate specificity of human Glycinamide ribonucleotide transformylase.
Archives of biochemistry and biophysics, 1999Co-Authors: Vincent D. Antle, Nathaniel Donat, Mei Hua, Pei Ling Liao, Robert Vince, Carol A CaperelliAbstract: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.
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substrate specificity of human Glycinamide ribonucleotide transformylase
Archives of Biochemistry and Biophysics, 1999Co-Authors: Vincent D. Antle, Nathaniel Donat, Mei Hua, Pei Ling Liao, Robert Vince, Carol A CaperelliAbstract: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.
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The Human Trifunctional Enzyme ofde NovoPurine Biosynthesis: Heterologous Expression, Purification, and Preliminary Characterization
Protein expression and purification, 1998Co-Authors: Mark T. Poch, Wen Qin, Carol A CaperelliAbstract: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.
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The Human Glycinamide Ribonucleotide Transformylase Domain: Purification, Characterization, and Kinetic Mechanism
Archives of biochemistry and biophysics, 1997Co-Authors: Carol A Caperelli, Eugene L. GirouxAbstract:Glycinamide ribonucleotide transformylase catalyzes the third reaction of de novo purine biosynthesis, namely, the conversion of Glycinamide ribonucleotide to N-formylGlycinamide ribonucleotide, with concomitant conversion of 10-formyltetrahydrofolate to tetrahydrofolate. This activity has been shown to be a target for cancer chemotherapy, which has generated renewed interest in both the enzyme and the pathway. Moreover, in higher eukaryotes this activity constitutes the C-terminal domain of a monomeric protein which also catalyzes two additional reactions of de novo purine biosynthesis. In this study, the human Glycinamide ribonucleotide transformylase domain has been expressed to high levels in Escherichia coli and purified to homogeneity. Our improved expression-purification system produces the desired activity exclusively in a soluble form and in higher abundance than previously achieved. The kinetic constants have been determined and the kinetic mechanism has been established as 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.
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Substrate Specificity of Glycinamide Ribonucleotide Synthetase from Chicken Liver
The Journal of biological chemistry, 1996Co-Authors: Vincent D. Antle, Carol A Caperelli, Mei Hua, Dashan Liu, B. Robert Mckellar, Robert VinceAbstract:Several analogs of Glycinamide ribonucleotide and phosphoribosylamine have been prepared and evaluated as substrates for Glycinamide ribonucleotide synthetase purified from chicken liver. Glycinamide ribonucleotide analogs include side chain modifications wherein the glycine side chain (R = CH2NH2) has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, ribose ring replacement by chiral cyclopentane and cyclopentene derivatives, and phosphate replacement by phosphonates. All of these, with the exception of the O-phosphonate, served as substrates for the reverse enzymatic reaction, with Vmax values comparable to that obtained with Glycinamide ribonucleotide, although the Km values ranged from 21 to 118 times the Km for Glycinamide ribonucleotide. Analogs of phosphoribosylamine examined as substrates for the forward reaction consist of chiral derivatives of cyclopentane and cyclopentene and a chiral carbocyclic phosphonate. These also served as substrates, with Km values ranging from 5 to 23 times the Km for phosphoribosylamine and with diminished Vmax values. These studies have begun to define the structural features of the nucleotide substrate necessary to support enzymatic activity. Sarcosine (N-methylglycine) and beta-alanine were also accepted as substrates, albeit with reduced affinity compared with glycine.
Meir Bialer - One of the best experts on this subject based on the ideXlab platform.
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Pharmacokinetic analysis and anticonvulsant activity of glycine and Glycinamide derivatives
Epilepsy research, 1999Co-Authors: Sherbel Sussan, Arie Dagan, Meir BialerAbstract:The objective of this study was to investigate the pharmacokinetics and pharmacodynamics (anticonvulsant activity and neurotoxicity) of a series of amide derivatives of Glycinamide in order to explore their structure pharmacokinetic-pharmacodynamic relationship and to discover a Glycinamide derivative which might have the potential to become a new antiepileptic agent. The following compounds were investigated: glycylglycine, glycylGlycinamide, gaboylGlycinamide, N-acetylglycine, N-acetylGlycinamide, N-acetylglycylGlycinamide, N-acetyl, N'-benzylGlycinamide, N-benzyloxycarbonylglycine or Z-glycine, Z-Glycinamide, Z-glycylglycine and Z-glycylGlycinamide. The anticonvulsant activity and neurotoxicity study was carried out in classical animal models for anticonvulsant screening. The pharmacokinetics of the active compounds was studied in dogs, which is a common animal model for a comparative crossover pharmacokinetic studies. Of the compounds investigated in this study, all the dipeptides of Glycinamide and the glycine derivatives were found to be inactive. The only two active compounds were: N-acetyl,N'-benzylGlycinamide (VII) and Z-Glycinamide (IX). These compounds demonstrated similar pharmacokinetic profiles. Unlike glycine or Glycinamide, compounds VII and IX, being lipophilic derivatives of Glycinamide, showed anticonvulsant activity in animal models due to their better pharmacodynamic and pharmacokinetic properties. The pharmacodynamics and pharmacokinetics of compounds VII and IX were similar to that of the potential new antiepileptics; N-valproylGlycinamide and phthaloylGlycinamide. This study provides certain clues concerning the structural requirements for the design of anticonvulsant-active glycine derivatives.
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pharmacokinetic analysis and antiepileptic activity of two new isomers of n valproyl Glycinamide
Biopharmaceutics & Drug Disposition, 1997Co-Authors: Salim Hadad, Meir BialerAbstract:: Valproyl Glycinamide (TV 1901-VPGD) is a new antiepileptic drug, which is currently undergoing clinical trials. The present study explored the pharmacokinetics and pharmacodynamics (anticonvulsant activity and neurotoxicity) of two new isomers of valproyl Glycinamide: valnoctyl Glycinamide (VCGD) and diisopropylacetyl (DIGD). Both VCGD and DIGD showed anticonvulsant activity and a safety margin in mice similar to those of VPGD. Following i.v. administration (556 mg) to six dogs, VCGD had a clearance (Cl) value of 3.8 +/- 1.1 Lh-1 (mean +/- SD), a volume of distribution (Vss) of 15 +/- 2 L, and a half-life (t1/2) of 1.9 +/- 0.3 h. DIGD had Cl, Vss, and t1/2 values of 10 +/- 0.8 Lh-1, 19 +/- 3 L, and 1.6 +/- 0.2 h, respectively. Neither VCGD nor DIGD operated as chemical drug delivery systems (CDDSs) of glycine, valnoctic acid, or diisopropyl acetic acid and both showed antiepileptic profiles different from that of valproic acid (VPA). Both Glycinamides were biotransformed to their glycine analogues with similar fractions metabolized (fm): 59 +/- 5% (VCGD) and 62 +/- 15% (DIGD). The two glycine metabolites, valnoctyl glycine (VCGA) and diisopropylacetyl glycine (DIGA), were also administered to the same dogs in order to calculate the above fm values. Both VCGA and DIGA had higher Cl and lower Vss values than VCGD and DIGD and therefore their mean t1/2 values were 0.43 +/- 0.02 and 0.30 +/- 0.07 h, respectively. VCGA and DIGA were excreted mainly intact in the urine, with fractions excreted unchanged (fe) of 60 +/- 9 and 55 +/- 7%, respectively. The improved pharmacokinetic profile of VCGD and DIGD relative to their glycine analogues may explain the similarity of their anticonvulsant activity to that of valproyl Glycinamide. The current study demonstrates the benefit of the structure-pharmacokinetic-pharmacodynamic relationship (SPPR) approach in developing and selecting a potent antiepileptic compound in intact animals based not only on its intrinsic pharmacodynamic activity but also on its improved pharmacokinetic profile.
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Pharmacokinetic analysis and antiepileptic activity of two new isomers of N‐valproyl Glycinamide
Biopharmaceutics & Drug Disposition, 1997Co-Authors: Salim Hadad, Meir BialerAbstract:: Valproyl Glycinamide (TV 1901-VPGD) is a new antiepileptic drug, which is currently undergoing clinical trials. The present study explored the pharmacokinetics and pharmacodynamics (anticonvulsant activity and neurotoxicity) of two new isomers of valproyl Glycinamide: valnoctyl Glycinamide (VCGD) and diisopropylacetyl (DIGD). Both VCGD and DIGD showed anticonvulsant activity and a safety margin in mice similar to those of VPGD. Following i.v. administration (556 mg) to six dogs, VCGD had a clearance (Cl) value of 3.8 +/- 1.1 Lh-1 (mean +/- SD), a volume of distribution (Vss) of 15 +/- 2 L, and a half-life (t1/2) of 1.9 +/- 0.3 h. DIGD had Cl, Vss, and t1/2 values of 10 +/- 0.8 Lh-1, 19 +/- 3 L, and 1.6 +/- 0.2 h, respectively. Neither VCGD nor DIGD operated as chemical drug delivery systems (CDDSs) of glycine, valnoctic acid, or diisopropyl acetic acid and both showed antiepileptic profiles different from that of valproic acid (VPA). Both Glycinamides were biotransformed to their glycine analogues with similar fractions metabolized (fm): 59 +/- 5% (VCGD) and 62 +/- 15% (DIGD). The two glycine metabolites, valnoctyl glycine (VCGA) and diisopropylacetyl glycine (DIGA), were also administered to the same dogs in order to calculate the above fm values. Both VCGA and DIGA had higher Cl and lower Vss values than VCGD and DIGD and therefore their mean t1/2 values were 0.43 +/- 0.02 and 0.30 +/- 0.07 h, respectively. VCGA and DIGA were excreted mainly intact in the urine, with fractions excreted unchanged (fe) of 60 +/- 9 and 55 +/- 7%, respectively. The improved pharmacokinetic profile of VCGD and DIGD relative to their glycine analogues may explain the similarity of their anticonvulsant activity to that of valproyl Glycinamide. The current study demonstrates the benefit of the structure-pharmacokinetic-pharmacodynamic relationship (SPPR) approach in developing and selecting a potent antiepileptic compound in intact animals based not only on its intrinsic pharmacodynamic activity but also on its improved pharmacokinetic profile.
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the disposition of valproyl Glycinamide and valproyl glycine in rats
Pharmaceutical Research, 1997Co-Authors: Simcha Blotnik, Felix Bergman, Meir BialerAbstract:Purpose. To investigate the disposition of valproyl Glycinamide and valproyl glycine in rats and to compare it with that of valproic acid (VPA) and valpromide which were studied previously.
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The Disposition of Valproyl Glycinamide and Valproyl Glycine in Rats
Pharmaceutical research, 1997Co-Authors: Simcha Blotnik, Felix Bergman, Meir BialerAbstract:To investigate the disposition of valproyl Glycinamide and valproyl glycine in rats and to compare it with that of valproic acid (VPA) and valpromide which were studied previously. The study was carried out by monitoring the brain and liver levels of valproyl Glycinamide and valproyl glycine (as a function of time after iv dosing) in addition to the regular pharmacokinetic (PK) monitoring of plasma and urine levels of these compounds. The following PK parameters were obtained for valproyl Glycinamide and valproyl glycine, respectively: clearance, 7.1 and 16 ml/ min/kg; volume of distribution (Vss), 0.78 and 0.41 l/kg; half-life, 1.1 and 0.37 h; and mean residence time, 1.8 and 0.4 h. The ratios of AUCs of valproyl Glycinamide of liver to plasma and brain to plasma were 0.70 and 0.66, respectively. The ratios of the AUCs of valproyl glycine of liver to plasma and brain to plasma were 0.19 and 0.02, respectively. Valproyl Glycinamide distributes better in the brain than VPA, a fact which may contribute to its better anticonvulsant activity. Valproyl glycine was barely distributed in the brain, a fact which may explain its lack of anticonvulsant activity. In addition to the liver, the brain was found to be a minor metabolic site of the biotransformation of valproyl Glycinamide to valproyl glycine.
Dale L. Boger - One of the best experts on this subject based on the ideXlab platform.
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Design, synthesis, and biological evaluation of simplified α-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase
Bioorganic & medicinal chemistry, 2003Co-Authors: Thomas H. Marsilje, Stephen J. Benkovic, Michael P. Hedrick, Joel Desharnais, Ali Tavassoli, Yan Zhang, Ian A. Wilson, Dale L. BogerAbstract:A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of Glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase).
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Design, synthesis, and biological evaluation of fluoronitrophenyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase.
Bioorganic & medicinal chemistry letters, 2000Co-Authors: Dale L. Boger, Jae Hoon Shim, Thomas H. Marsilje, Qing Jin, Michael P. Hedrick, Stephen J. Baker, René A. Castro, Stephen J. BenkovicAbstract:The examination results of a novel series of potential inhibitors of Glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. These agents incorporate an electrophilic fluoronitrophenyl group that can potentially react with an active site nucleophile or the substrate GAR/AICAR amine via nucleophilic aromatic substitution.
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Conformationally restricted analogues designed for selective inhibition of GAR Tfase versus thymidylate synthase or dihydrofolate reductase.
Bioorganic & medicinal chemistry, 2000Co-Authors: Dale L. Boger, Jae Hoon Shim, Marc A. Labroli, Thomas H. Marsilje, Qing Jin, Michael P. Hedrick, Stephen J. Baker, Stephen J. BenkovicAbstract:The synthesis and evaluation of a series of conformationally restricted analogues of 10-formyl-tetrahydrofolate as potential inhibitors of Glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide transformylase (AICAR Tfase) are reported.
Robert Vince - One of the best experts on this subject based on the ideXlab platform.
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Substrate specificity of human Glycinamide ribonucleotide transformylase.
Archives of biochemistry and biophysics, 1999Co-Authors: Vincent D. Antle, Nathaniel Donat, Mei Hua, Pei Ling Liao, Robert Vince, Carol A CaperelliAbstract: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.
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substrate specificity of human Glycinamide ribonucleotide transformylase
Archives of Biochemistry and Biophysics, 1999Co-Authors: Vincent D. Antle, Nathaniel Donat, Mei Hua, Pei Ling Liao, Robert Vince, Carol A CaperelliAbstract: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.
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Substrate Specificity of Glycinamide Ribonucleotide Synthetase from Chicken Liver
The Journal of biological chemistry, 1996Co-Authors: Vincent D. Antle, Carol A Caperelli, Mei Hua, Dashan Liu, B. Robert Mckellar, Robert VinceAbstract:Several analogs of Glycinamide ribonucleotide and phosphoribosylamine have been prepared and evaluated as substrates for Glycinamide ribonucleotide synthetase purified from chicken liver. Glycinamide ribonucleotide analogs include side chain modifications wherein the glycine side chain (R = CH2NH2) has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, ribose ring replacement by chiral cyclopentane and cyclopentene derivatives, and phosphate replacement by phosphonates. All of these, with the exception of the O-phosphonate, served as substrates for the reverse enzymatic reaction, with Vmax values comparable to that obtained with Glycinamide ribonucleotide, although the Km values ranged from 21 to 118 times the Km for Glycinamide ribonucleotide. Analogs of phosphoribosylamine examined as substrates for the forward reaction consist of chiral derivatives of cyclopentane and cyclopentene and a chiral carbocyclic phosphonate. These also served as substrates, with Km values ranging from 5 to 23 times the Km for phosphoribosylamine and with diminished Vmax values. These studies have begun to define the structural features of the nucleotide substrate necessary to support enzymatic activity. Sarcosine (N-methylglycine) and beta-alanine were also accepted as substrates, albeit with reduced affinity compared with glycine.
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Substrate Specificity of Glycinamide Ribonucleotide Transformylase from Chicken Liver
The Journal of biological chemistry, 1996Co-Authors: Vincent D. Antle, Carol A Caperelli, Mei Hua, Dashan Liu, B. Robert Mckellar, Robert VinceAbstract:Several Glycinamide ribonucleotide analogs have been prepared and evaluated as substrates and/or inhibitors of Glycinamide ribonucleotide transformylase from chicken liver. The side chain modified analogs, in which the glycine side chain, R = CH2NH2, has been replaced by R = CH2NHCH3 and R = CH2CH2NH2, are substrates, with V/K (relative intensity) of 2.4% and 16.3%, respectively. Several carbocyclic analogs of Glycinamide ribonucleotide, including the phosphonate derivative of carbocyclic Glycinamide ribonucleotide, did not serve as substrates, but were inhibitors of the enzyme, competitive against Glycinamide ribonucleotide, with Ki values ranging from 7.4 to 23.6 times the Km for Glycinamide ribonucleotide. However, the O-phosphonate analog of carbocyclic Glycinamide ribonucleotide did support enzymatic activity, with V/K (relative intensity) of 0.8%. In addition, Glycinamide ribonucleoside was neither a substrate for, nor an inhibitor of, Glycinamide ribonucleotide transformylase. Furthermore, alpha-Glycinamide ribonucleotide had no effect on enzyme activity. These studies have begun to define the structural features of the nucleotide substrate required to support enzymatic activity.