Biomedical subjects

F Maley

Publications and source records attributed to F Maley.

At least 37 records · Page 2Linked to original sources

PubMed1997

Intracellular location of thymidylate synthase and its state of phosphorylation.

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5, 10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50-100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.

Animals↗

PubMed1997

Use of strain in a stereospecific catalytic mechanism: crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate.

Two crystal structures for E. coli thymidylate synthase (TS) bound to the mechanism-based inhibitor 5-fluoro-dUMP (FdUMP) and methylenetetrahydrofolate (CH2THF) have been determined to 2.6 and 2.2 A nominal resolutions, with crystallographic R factors of 0.180 and 0.178, respectively. The inhibitor and cofactor are well ordered in both structures and display covalent links to each other and to Cys 146 in the TS active site. The structures are in general agreement with a previous report for this complex (D. A. Matthews et al. (1990) J. Mol. Biol. 214, 937-948), but differ in two key respects: (i) the methylene bridge linking FdUMP and CH2THF is rotated about 60 degrees to a different position and (ii) the electron density for C6 of FdUMP, which is covalently linked to Cys 146, is more diffuse than for the other atoms in the pyrimidine ring. The ligand arrangement observed in the previous structure led the authors to propose that a large conformational change in ligand geometry must occur in order to facilitate catalysis and yield the correct chirality in the methyl of product dTMP. The new structures suggest a different mechanism for product formation that does not require ligands to greatly alter their conformations during catalysis and which makes use of instability in the nucleotide-Cys 146 thiol adduct to avoid a deep free energy well and assist in proton abstraction from dUMP. All intermediates in the proposed mechanism were modeled and energy minimized in the TS active site, and all can be accommodated in the present structures. The role of ligand-induced conformational change in the TS mechanism and the possibility of Tyr 94 acting as a base during catalysis are also discussed.

Catalysis↗

PubMed1997

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu.

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.

Antineoplastic Agents↗

PubMed1997

High-level expression of human thymidylate synthase.

A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli. By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.

Escherichia coli↗

PubMed1996

Protein-protein interactions involving T4 phage-coded deoxycytidylate deaminase and thymidylate synthase.

The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.

Bacteriophage T4↗

PubMed1996

Thermodynamic stabilization of nucleotide binding to thymidylate synthase by a potent benzoquinazoline folate analogue inhibitor.

The stabilization of dUMP, FdUMP, and dGMP binding to Escherichia coli thymidylate synthase (TS) in the presence and absence of a folate analogue inhibitor of TS, 1843U, was determined by differential scanning calorimetry. When the enzyme is thermally unfolded in the presence of dUMP, two separate temperature transitions are evident, although only one binding site/dimer was detected in equilibrium dialysis experiments. In the absence of dUMP, TS shows a major peak of unfolding at 45 degrees C with a shoulder at 47 degrees C. In the presence of increasing amounts of dUMP progressive changes in the size of each peak occur, each associated with a higher temperature of unfolding. At a ratio of dUMP/TS of 100, a major peak predominates with an unfolding temperature (Td) of 60 degrees C. FdUMP shows a similar profile, while dGMP does not alter the Td of the enzyme since dGMP alone does not bind to TS. Despite the fact that 1843U binds tightly to TS in the absence of nucleotide ligands [Dev, I. K., Dallas, W.S., Ferone, R., Hanlon, H., McKee, D.D., & Yates, B. B. (1994) J.Biol. Chem. 269, 1873-1882], it exhibits only a small effect on the Td profile of TS. However, when 1843U is present, in addition to the nucleotides (dUMP, FdUMP, or dGMP), a Td of 72 degrees C is achieved and the enthalpy of unfolding is increased by one-third. The stabilizing effect of substrate binding to TS by 1843U examined by thermodynamic parameters can be attributed to the considerable extra amount of free energy released on formation of the ternary complex of TS-1843U-nucleotide. The tightness of this complex is due to the stacking energy that results from Van der Waals contacts between the nucleotide purine or pyrimidine ring and the benzoquinazoline ring of 1843U [Weichsel, A., Montfort, W. R., Cieśla, J., & Maley, F. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3493-3497], which induces a local conformational change in the protein. This conformational change is associated with a significant positive entropy change, which suggests that water is expelled from the active site region.

Calorimetry, Differential Scanning↗

PubMed1996

Studies on identifying the catalytic role of Glu-204 in the active site of yeast invertase.

In a previous study on yeast invertase (Reddy, A., and Maley, F. (1990) J. Biol. Chem. 265, 10817-10820), we identified Asp-23 through the procedures of affinity labeling and site-directed mutagenesis as a catalytic nucleophile. In the present study we undertook to determine other residues involved in the catalytic process. Earlier studies suggested histidine as a potential proton donor in the hydrolysis of sucrose, but by mutagenizing each of the enzyme's four histidines this amino acid was eliminated from consideration. Another candidate appeared to be cysteine, since iodine at about a 2-fold molar excess inactivated invertase by modifying both of the enzyme's cysteine residues. Dithiothreitol treatment restored the sulfhydryl groups and enzyme activity. Replacement of each of the cysteines with alanines revealed that C108A invertase retained full activity whereas C205A was reduced about 4-fold in its kcat. A comparison of the amino acid sequences of fructosylhydrolases revealed a conserved region coincident with Glu-204/Cys-205. Mutagenizing Glu-204 to Ala resulted in a 3, 000-fold reduction in the kcat of invertase indicating that Glu-204 plays a major role in catalysis. Based on these findings, a mechanism is proposed for the hydrolysis of sucrose which involves Asp-23 as a nucleophile and Glu-204 as an acid/base catalyst.

Amino Acid Sequence↗

PubMed1995

Chromosomal location and structural organization of the human deoxycytidylate deaminase gene.

Deoxycytidylate deaminase is an allosteric enzyme whose impairment can lead to deoxynucleotide imbalances that affect the fidelity of DNA synthesis. A DNA fragment encompassing the gene for deoxycytidylate deaminase has been isolated from a human lung fibroblast genomic library and sequenced in both directions through 26,764 base pairs. The previously isolated cDNA, which was used to establish the amino acid sequence for this enzyme (Weiner, K.X.B., Weiner, R.S., Maley, F., and Maley, G.F. (1993) J. Biol. Chem. 268, 12983-12989) was instrumental in isolating this gene. The gene consists of five exons of about 100 base pairs each, separated by four introns. The most striking feature of the genomic structure is that the second and third exons are separated by an intron of about 20 kilobases. The chromosomal location of the deaminase gene was determined by fluorescence in situ hybridization as 4q35, which is the extreme end of this chromosome. The position of this gene on chromosome 4, in addition to the role of its product in limiting potentially detrimental mutations, suggests that the normal operation of both the gene and its product is important to the well being of the organism.

Base Sequence↗

PubMed1995

Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

A folate analogue, 1843U89 (U89), with potential as a chemotherapeutic agent due to its potent and specific inhibition of thymidylate synthase (TS; EC 2.1.1.45), greatly enhances not only the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and dUMP to Escherichia coli TS but also that of dGMP, GMP, dIMP, and IMP. Guanine nucleotide binding was first detected by CD analysis, which revealed a unique spectrum for the TS-dGMP-U89 ternary complex. The quantitative binding of dGMP relative to GMP, FdUMP, and dUMP was determined in the presence and absence of U89 by ultrafiltration analysis, which revealed that although the binding of GMP and dGMP could not be detected in the absence of U89 both were bound in its presence. The Kd for dGMP was about the same as that for dUMP and FdUMP, with binding of the latter two nucleotides being increased by two orders of magnitude by U89. An explanation for the binding of dGMP was provided by x-ray diffraction studies that revealed an extensive stacking interaction between the guanine of dGMP and the benzoquinazoline ring of U89 and hydrogen bonds similar to those involved in dUMP binding. In addition, binding energy was provided through a water molecule that formed hydrogen bonds to both N7 of dGMP and the hydroxyl of Tyr-94. Accommodation of the larger dGMP molecule was accomplished through a distortion of the active site and a shift of the deoxyribose moiety to a new position. These rearrangements also enabled the binding of GMP to occur by creating a pocket for the ribose 2' hydroxyl group, overcoming the normal TS discrimination against nucleotides containing the 2' hydroxyl.

Circular Dichroism↗

PubMed1995

Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases.

Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.

Amino Acid Sequence↗

PubMed1995

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.

Base Sequence↗

PubMed1995

Complete restoration of activity to inactive mutants of Escherichia coli thymidylate synthase: evidence that E. coli thymidylate synthase is a half-the-sites activity enzyme.

Escherichia coli thymidylate synthase (TS) is a dimeric protein containing identical subunits. When R126E, an inactive mutant of this enzyme, was incubated at room temperature with other inactive mutants of E. coli, TS enzyme activity gradually reappeared. The rate of activity restoration was dependent on the mutant employed. In the case of C146W, an active site mutant, the half-time required for maximal activity restoration was about one hour, which was about 500-fold faster than that obtained with C146S. The final specific activity of the mutant mixtures, based on the concentration of R126E, was equivalent to that of the wild-type TS (WT-TS). However, when the activities of E. coli WT-TS and mutant TS mixtures were compared for their extents of renaturation following denaturation as described for Lactobacillus casei TS [Pookanjanatavip, M., Yuthavong, Y., Greene, P. J., & Santi, D. V. (1992) Biochemistry 31, 10303-10309], only about one-half of the activity of WT-TS was restored, implying that the denaturation-renaturation procedure was less efficient than allowing the native TS mutant dimers to exchange subunits. If, as proposed, subunit exchange is responsible for the observed restoration of activity to the E. coli mutant TS mixtures, it would suggest that only one active site cysteine, that provided by R126E in the dimer (R126E)-(C146W), is sufficient to yield the same kcat as WT-TS, which contains one active site cysteine in each subunit. Other mutant dimers that contain both active site cysteines such as (R126E)-(Y94A) and (R126E)-(I264Am) are also fully active, even though one of the subunits is functionally inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1995

An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.

Numerous reports describe the phage T4 enzymes thymidylate synthase and dihydrofolate reductase as structural components of the baseplate. However, Y. Wang and C. K. Mathews (J. Virol. 63:4736-4743, 1989) reported that antisera against the respective recombinant enzymes failed to neutralize phage infectivity, in contrast to previous results. Moreover, a deletion mutant lacking the genes for these two enzymes adsorbed normally to host cells. Since these findings tended to undermine the idea of the two enzymes as structural proteins, we developed a quantitative immunoblot assay to resolve the issue directly. Our results show that both enzymes are present only as minor contaminants (< 0.05 copy per phage) and as such cannot be bona fide structural proteins.

Bacteriophage T4↗

PubMed1995

Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro.

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.

Base Sequence↗

PubMed1994

Crystal structure of thymidylate synthase from T4 phage: component of a deoxynucleoside triphosphate-synthesizing complex.

Thymidylate synthase from phage T4 (T4TS) is part of a complex of several enzymes required for coordinate DNA synthesis in infected Escherichia coli cells. It has been proposed that similar complexes of enzymes related to DNA synthesis are also functional in eukaryotes [Pardee, A. B. (1989) Science 246, 603-608]. To delineate the role of structure in the function of this complex, we have solved the structure of T4TS as a basis for mapping the complex by mutagenesis. The 3.1 A structure of the unliganded enzyme was determined by molecular replacement and refined to 19.9% for all data. Three inserts and one deletion in the coding region are unique to T4TS, and all sites lie on one side of the enzyme surface, possibly encoding unique T4 specific intermolecular interactions during the infective cycle. The crystal structure is generally in the open, unliganded conformation seen in unliganded E. coli TS, as opposed to the closed, ternary complex conformation, except that the critically important C-terminus is inserted into the active site hydrogen bonded to residue Asn85, as seen in functional ternary complex structures. Other differences between E. coli TS and T4TS appear to explain the enhanced binding of folyl polyglutamate to the latter.

Amino Acid Sequence↗

PubMed1994

The effect of reducing reagents on binding of thymidylate synthase protein to thymidylate synthase messenger RNA.

Human thymidylate synthase (TS) protein specifically binds to its own TS mRNA and functions as a translational repressor. In the presence of reducing agents, the RNA binding activity of TS protein is significantly enhanced. In contrast, treatment of TS protein with the oxidizing agent diamide inhibits RNA binding. Scatchard analysis reveals that in the presence of the reducing agent 2-mercaptoethanol, the TS protein/TS mRNA interaction changes from low (Kd = 66 nM) to high (Kd = 2.6 nM) apparent affinity. The catalytic activity of TS is increased by up to 6.5-fold in the presence of 2-mercaptoethanol. These studies demonstrate that the interaction between TS protein and its target TS mRNA is sensitive to the presence of reducing reagents and is dependent upon a reversible sulfhydryl switch mechanism.

Binding Sites↗

PubMed1994

Identification of a site necessary for allosteric regulation in T4-phage deoxycytidylate deaminase.

An allosteric inhibitor of dCMP deaminase, dTTP, forms a photolabile covalent bond with T4-phage dCMP deaminase in the presence of UV light at 254 nm. The importance of the methyl group in this process is supported by the findings that dUTP, also an allosteric inhibitor, does not photofix to the enzyme and that tritium is released from [methyl-3H dTTP during the course of the photofixation. That the bond formed is photolabile is demonstrated by the fact that tritium is released by about 10-fold over the amount of nucleotide that is photofixed. The amino acid that covalently binds dTTP in T4-dCMP deaminase was identified as Phe112. On conversion of Phe112 to an alanine by site-directed mutagenesis, there was a dramatic change in the enzyme's response to its allosteric effectors when measured early in the reaction, in that the mutant enzyme was as active as the wild-type even in the absence of dCTP and was only weakly inhibited by dTTP. However, after 10-15% of the substrate had been deaminated, the reaction rate fell off rather markedly, indicating either that an inhibitor was being accumulated on the enzyme or that the enzyme was being irreversibly inactivated with time. That the latter was not the case was shown by the addition of dCTP to the reaction, which restored the rate to that expected when it was present initially. Furthermore, we showed that, consistent with the observed loss of allosteric regulation by dCTP and dTTP, the affinity of the mutant enzyme for dTTP and dCTP as determined by binding studies was greatly reduced relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1994

Water-mediated substrate/product discrimination: the product complex of thymidylate synthase at 1.83 A.

In an irreversible enzyme-catalyzed reaction, strong binding of the products would lead to substantial product inhibition. The X-ray crystal structure of the product complex of thymidylate synthase (1.83-A resolution, R factor = 0.183 for all data between 7.0 and 1.83 A) identifies a bound water molecule that serves to disfavor binding of the product nucleotide, dTMP. This water molecule is hydrogen bonded to absolutely conserved Tyr 146 (using the Lactobacillus casei numbering system) and is displaced by the C7 methyl group of the reaction product thymidylate. The relation between this observation and kinetic and thermodynamic values is discussed. The structure reveals a carbamate modified N-terminus that binds in a highly conserved site, replaced by side chains that can exploit the same site in other TS sequences. The enzyme-products complex is compared to the previously determined structure of enzyme-substrate-cofactor analog. This comparison reveals changes that occur between the first covalent complex formed between enzyme and substrate with an inhibitory cofactor analog and the completed reaction. The almost identical arrangement of ligands in these two structures contributes to our model for the TS reaction and verifies the physiological relevance of the mode in which potent inhibitors bind to this target for rational drug design.

Carbamates↗