DrugBank:EXPT00572 - FACTA Search

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Query: DrugBank:EXPT00572 (Asn)
11,732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The strictly conserved residue leucine-54 of Escherichia coli dihydrofolate reductase forms part of the hydrophobic wall which binds the p-aminobenzoyl side chain of dihydrofolate. In addition to the previously reported glycine-54 mutant, isoleucine-54 and asparagine-54 substitutions have been constructed and characterized with regard to their effects on binding and catalysis. NADP+ and NADPH binding is virtually unaffected with the exception of a 15-fold decrease in NADPH dissociation from the Gly-54 mutant. The synergistic effect of NADPH on tetrahydrofolate dissociation seen in the wild-type enzyme is lost in the isoleucine-54 mutant: little acceleration is seen in tetrahydrofolate dissociation when cofactor is bound, and there is no discrimination between reduced and oxidized cofactor. The dissociation constants for dihydrofolate and methotrexate increase in the order Leu less than Ile less than Asn less than Gly, varying by a maximum factor of 1700 for dihydrofolate and 6300 for methotrexate. Despite these large changes in binding affinity, the hydride transfer rate of 950 s-1 in the wild-type enzyme is decreased by a constant factor of ca. 30 (2 kcal/mol) regardless of the mutant. Thus, the contributions of residue 54 to binding and catalysis appear to have been separated.
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PMID:Hydrophobic interactions via mutants of Escherichia coli dihydrofolate reductase: separation of binding and catalysis. 266 66

The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the pyrimidine ring of the substrate dUMP. The carboxamide moiety forms a cyclic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of mutants at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants were catalytically active. Steady-state kinetic parameters of N229 mutants varied 17- and 115-fold in the Km values for 5,10-methylene-5,6,7,8-tetrahydrofolate and dUMP, respectively, 1000-fold in kcat values, and 10,000-fold in kcat/Km values. Wild-type TS possesses lower Km and higher kcat and kcat/Km values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential for, binding and catalysis. When the wild-type enzyme was not considered, there were excellent correlations between log kcat and the hydrophobicity of the side chains at position 229, in which the more hydrophobic side chains showed higher values. Our results suggest a unique interaction between N229 and the substrates that seems important in appropriately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229, the electrophilic catalyst that transfers protons to the O-4 and stabilizes enol intermediates is a highly conserved molecule of water.
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PMID:Asparagine 229 in thymidylate synthase contributes to, but is not essential for, catalysis. 837 36

The crystal structure of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) as a binary complex with folinic acid (5-formyl-5,6,7,8-tetrahydrofolate; also called leucovorin or citrovorum factor) has been solved in two space groups, P6(1) and P6(5), with, respectively, two molecules and one molecule per asymmetric unit. The crystal structures have been refined to an R-factor of 14.2% at resolutions of 2.0 and 1.9 A. The P6(1) structure is isomorphous with several previously reported ecDHFR binary complexes [Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662; Reyes, V.M., Sawaya, M.R., Brown, K.A., & Kraut, J. (1995) Biochemistry 34, 2710-2723]; enzyme and ligand conformations are very similar to the P6(1) 5,10-dideazatetrahydrofolate complex. While the two enzyme subdomains of the P6(1) structure are nearly in the closed conformation, exemplified by the methotrexate P6(1) binary complex, in the P6(5) structure they are in an intermediate conformation, halfway between the closed and the fully open conformation of the apoenzyme [Bystroff, C., Oatley, S.J., & Kraut, J. (1990) Biochemistry 29, 3263-3277]. Thus crystal packing strongly influences this aspect of the enzyme structure. In contrast to the P6(1) structure, in which the Met-20 loop (residues 9-23) is turned away from the substrate binding pocket, in the P6(5) structure the Met-20 loop blocks the pocket and protrudes into the cofactor binding site. In this respect, the P6(5) structure is unique. Additionally, positioning of a Ca2+ ion (a component of the crystallization medium) is different in the two crystal packings: in the P6(1) structure it lies at the boundary between the two molecules of the asymmetric unit, while in P6(5) it coordinates two water molecules, the hydroxyl group of an ethanol molecule, and the backbone carbonyl oxygens of Glu-17, Asn-18, and Met-20. The Ca2+ ion thus stabilizes a single turn of 3(10) helix (residues 16-18 in the Met-20 loop), a second unique feature of the P6(5) crystal structure. The disposition of the N5-formyl group in these structures indicates formation, at least half of the time, of an intramolecular hydrogen bond between the formyl oxygen and O4 of the tetrahydropterin ring. This observation is consistent with the existence of an enol-keto equilibrium in which the enolic tautomer is favored when a hydrogen-bond acceptor is present between O4 and N5. Such would be the case whenever a water molecule occupies that site as part of a hypothetical proton-relay mechanism. Two arginine side chains, Arg-52 in the P6(5) structure and Arg-44 in molecule A of the P6(1) structure, are turned away drastically from the ligand (p-aminobenzoyl)glutamic acid moiety as compared with previously reported DHFR binary complex structures. As in the ecDHFR dideazatetrahydrofolate complex, in both the P6(1) and P6(5) structures a water molecule bridges pteridine O4 and Trp-22(N epsilon 1) with ideal geometry for hydrogen bonding, perhaps contributing to the slow release of 5,6,7,8-tetrahydrofolate from the enzyme-product complex. When either the P6(1) or the P6(5) structures are superimposed with the NADPH holoenzyme [Sawaya, M. R. (1994) Ph.D. Dissertation, University of California, San Diego], we find that the distances between the nicotinamide C4 and pteridine C6 and C7 are very short, 2.1 and 1.7 A in the P6(1) case and 2.0 and 1.4 A in the P6(5) case, perhaps in part explaining the more rapid release of tetrahydrofolate from the enzyme-product complex when NADPH is bound.
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PMID:Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: evidence for enolization of pteridine O4. 867 26

C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism. In this study, we have examined the role of a highly conserved aspartate residue, Asp449 of yeast cytoplasmic C1-THF synthase. Asp449 is part of a putative folate binding site found in many proteins that bind 10-formyl-THF. The corresponding aspartate has been identified as a critical catalytic residue in Escherichia coli and human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer. In order to determine if Asp449 has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449 of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala. The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability. All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449 is not a critical catalytic residue. However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site. Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes.
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PMID:Site-directed mutagenesis of a highly conserved aspartate in the putative 10-formyl-tetrahydrofolate binding site of yeast C1-tetrahydrofolate synthase. 880 78

A rabbit liver cDNA library in phage lambdagt10 was screened using the coding cDNA for human cytosolic serine hydroxymethyltransferase. A clone of 1754 bp was isolated and the nucleotide sequence showed an open reading frame of 1455 bp, which coded for rabbit cytosolic serine hydroxymethyltransferase and was flanked by 12 bp at the 5' end and 287 bp at the 3' end. The full-length cDNA was then cloned into a pET22b vector as a NdeI-EcoRI insert. HMS174(DE3) cells were transformed with this plasmid and, after induction with isopropyl beta-D-thiogalactopyranoside, expressed a catalytically active serine hydroxymethyltransferase. The enzyme was purified and shown to be the expressed rabbit enzyme lacking the first methionine residue. Spectral characteristics of the bound pyridoxal phosphate and kinetic constants for the natural substrates L-serine and tetrahydrofolate were essentially identical to the values obtained previously for the rabbit cytosolic enzyme. The pattern of bands shown by the pure recombinant enzyme on an isoelectric focusing gel containing 6 M urea showed a major band and a minor band representing about 15-20% of the protein. Upon incubation of the recombinant enzyme at pH 7.3 and 37 degreesC, three new bands were observed on isoelectric focusing with the concomitant formation of isoaspartyl residues, as determined by reactivity with protein isoaspartyl methyltransferase. These results are consistent with deamidation of Asn residues to isoaspartyl during the in vitro incubation. The enzyme purified from rabbit liver has previously been shown to contain isoaspartyl residues.
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PMID:Purification and characterization of recombinant rabbit cytosolic serine hydroxymethyltransferase. 967 60

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.
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PMID:D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine. 975 79

To probe the role of cation-pi and amino-pi interactions in the context of protein-ligand interactions, the stability of 55 X-ray cation/amino-pi motifs involving the Ade moieties of cofactor molecules and Arg, Lys, Asn, or Gln side chains of their host protein was evaluated using quantum chemistry calculations. The conjunction of vacuum interaction energies, vibrational entropy, and solvation contributions led to identify Arg-Ade as the most favorable cation/amino-pi complex in the solvents considered, followed by Asn/Gln-Ade and Lys-Ade: their minimum interaction free energies are approximately equal to -7, -4, and -2 kcal/mol, respectively, in the solvents of dielectric constant similar to that estimated for proteins (i.e., acetone, THF, and CCl(4)). Remarkably, these free-energy values of cation/amino-pi interactions correlate well with their frequency of occurrences in protein-ligand structures, which corroborates our approach in the absence of experimental data.
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PMID:Free-energy calculations of protein-ligand cation-pi and amino-pi interactions: from vacuum to proteinlike environments. 1461 Dec 35

Serine hydroxymethyltransferase (SHMT) is a key enzyme in the formation and regulation of the folate one-carbon pool. Recent studies on human subjects have shown the existence of two single nucleotide polymorphisms that may be associated with several disease states. One of these mutations results in Ser394 being converted to an Asn (S394N) and the other in the change of Leu474 to a Phe (L474F). These mutations were introduced into the cDNA for both human and rabbit cytosolic SHMT and the mutant enzymes expressed and purified from an Escherichia coli expression system. The mutant enzymes show normal values for kcat and Km for serine. However, the S394N mutant enzyme has increased dissociation constant values for both glycine and tetrahydrofolate (tetrahydropteroylglutamate) and its pentaglutamate form compared to wild-type enzyme. The L474F mutant shows lowered affinity (increased dissociation constant) for only the pentaglutamate form of the folate ligand. Both mutations result in decreased rates of pyridoxal phosphate addition to the mutant apo enzymes to form the active holo enzymes. Neither mutation significantly affects the stability of SHMT or the rate at which it converts 5,10-methenyl tetrahydropteroyl pentaglutamate to 5-formyl tetrahydropteroyl pentaglutamate. Analysis of the structures of rabbit and human SHMT show how mutations at these two sites can result in the observed functional differences.
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PMID:Properties of human and rabbit cytosolic serine hydroxymethyltransferase are changed by single nucleotide polymorphic mutations. 1613 37

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.
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PMID:Modular organization of FDH: Exploring the basis of hydrolase catalysis. 1659 35

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THF. SHMT also catalyses several THF-independent side reactions such as cleavage of beta-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect on either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.
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PMID:Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding. 1904 38

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