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R B Dunlap

Publications and source records attributed to R B Dunlap.

At least 19 recordsLinked to original sources
PubMed1992

Selenomethionyl dihydrofolate reductase from Escherichia coli. Comparative biochemistry and 77Se nuclear magnetic resonance spectroscopy.

The biosynthetic replacement of Met residues by selenomethionine (SeMet) facilitates the determination of three-dimensional structure by multiwavelength anomalous diffraction (Yang, W., Hendrickson, W. A., Crouch, R.J., and Satow, Y. (1990) Science 249, 1398-1405). In an effort to examine any biochemical effects due to the replacement of Met residues by SeMet, we chose to compare the kinetic and binding properties of selenomethionyl dihydrofolate reductase with those of the wt enzyme. There are 5 Met residues in Escherichia coli dihydrofolate reductase with 2 located in the Met-20 loop, which is a sequence of residues forming a lid over the active site. Utilizing plasmid pWT8, which affords 10-15% soluble protein as E. coli dihydrofolate reductase, we readily isolated both the SeMet and wt enzymes from E. coli DL41 utilizing a novel purification protocol. Both enzymes exhibited essentially the same kinetic and binding properties, including specific activities (45 mumol/min/mg), Km (7,8-dihydrofolate = 0.39 microM; NADPH = 2.0 microM), kcat (13.5/s), and 1:1 noncovalent inhibitory binding ratios with methotrexate. The inhibitory effects of divalent and monovalent cations on activity were also assessed, with the SeMet-containing enzyme exhibiting a uniformly greater sensitivity than the wt enzyme. We conclude that the biochemical properties of dihydrofolate reductase are virtually unperturbed by SeMet inclusion. Analysis of SeMet dihydrofolate reductase by 77Se nuclear magnetic resonance spectroscopy revealed five distinct resonances, thus indicating the potential value of this technique in employing selenium as a nonperturbing NMR probe of protein structure and function.

Cations, Divalent↗
PubMed1992

Muscarinic and nicotinic activities of the novel acetylcholine analog acetylselenonium choline.

The present paper further characterizes the cholinergic properties of acetylselenonium choline (ASeCh, (CH3)2Se+CH2CH2OCOCH3). The data demonstrate that ASeCh possesses muscarinic receptor agonist properties as evidenced by vasodepressor and smooth muscle contractile activities which are enhanced by physostigmine and antagonized by atropine. ASeCh also possessed nicotinic agonist activity on frog rectus abdominis tissue which was potentiated by physostigmine, and blocked by d-tubocurarine. The relative potencies of ASeCh ranged from approximately 1% to approximately 6% of the potency of acetylcholine in the three types of preparations examined.

Acetylcholine↗
PubMed1992

Selected biological activities of novel selenonium choline analogs.

1. The novel choline analogs selenonium choline (SeCh) and acetylselenonium choline (ASeCh) have been examined for selected biological activities. 2. ASeCh was found to be an alternative substrate for acetylcholine esterase with Km and Vmax values similar to acetylcholine. 3. ASeCh and SeCh inhibited acetylthiocholine hydrolysis by acetylcholinesterase with IC50 values similar to acetylcholine and choline. 4. SeCh exerted a protective action against physostigmine and DFP induced toxicity. 5. SeCh (85 mg/kg) was found to be 3 times more toxic in mice than choline.

Acetylcholine↗
PubMed1992

Catalysis and ligand binding by thymidylate synthase immobilized on thiopropyl-sepharose 6B.

Lactobacillus casei thymidylate synthase, which employs the thiol of cysteine-198 as a covalent catalyst, was reversibly coupled to thiopropyl-Sepharose 6B through the catalytic sulfhydryl group of one of its two subunits, yielding an immobilized heterodimeric form of the enzyme possessing one free active site and one covalently modified active site. Enzyme inactivated by treatment with N-ethylmaleimide, which selectively modifies the active site cysteines but not the remaining cysteines (cys-244), failed to react with the resin. Modified assay procedures were developed and utilized to characterize the activity and ligand binding properties of this unique enzymic species. The immobilized enzyme was found to catalyze thymidylate formation at its free active site but exhibited lowered specific activity (13-fold) and kcat (16-fold) values and an increased Km for dUMP (4-fold) when compared to the native, soluble enzyme. Immobilized enzyme also formed a covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate with a stoichiometry of 0.32 (mol FdUMP: mol enzyme), about half the predicted value of 0.6-0.7. The results of this initial study suggest that the active sites of the native enzyme dimer are asymmetrical in nature, and that the chemical status of the catalytic sulfhydryl groups may play a key role in directing communication between the subunits.

Enzymes, Immobilized↗
PubMed1991

Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme.

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of recombinant mouse thymidylate synthase.

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

Animals↗
PubMed1990

Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase.

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.

Buffers↗
PubMed1990

Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase.

The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.

Bacterial Proteins↗
PubMed1990

Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding.

The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.

Amino Acid Sequence↗
PubMed1989

Efficient synthesis of mouse thymidylate synthase in Escherichia coli.

The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3)[pLysS]. When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low. When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein. The recombinant enzyme was purified to homogeneity by affinity chromatography. The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts. Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts. The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques.

Animals↗
PubMed1989

A rapid, reliable, and convenient method for the purification of thymidylate synthase from amethopterin-resistant Lactobacillus casei.

The results presented herein describe the development of a new method for the purification of thymidylate synthase from amethopterin-resistant L. casei. This method includes three chromatographic steps: batchwise CM-Sephadex, Q-Sepharose, and 10-formylfolate-Sepharose affinity steps, and the whole procedure can be performed in three days. Additionally, the procedure has consistently produced enzyme with a specific activity of 3.0 to 3.2. The first step, the batchwise purification of L. casei cell free extracts on CM-Sephadex, resulted in a 120-fold purification with 115% recovery of the activity applied to the resin. The Q-Sepharose purification step yielded a 1.5-fold purification producing enzyme with a specific activity of 2.4 units/mg. As the final step in the sequence, 10-formylfolate-Sepharose affinity chromatography yielded enzyme with a specific activity of 3.0-3.2 units/mg. The entire procedure provided a 230-fold purification with 67% recovery of the activity originally present in the cell free extract. However, the purified enzyme required extensive dialysis to achieve maximal activity. The data presented also clearly demonstrate the resolution of thymidylate synthase on the 10-formylfolate affinity column into forms which have different specific activity values and FdUMP binding ratios. Generally, enzyme with low activity leaches off this affinity matrix first, while enzyme with high activity elutes off last. These results are in accord with the sensitivity of the dimeric thymidylate synthase active site cysteines to oxidization. Consequently, when one of the two active site cysteines is oxidized, the enzyme loses the ability to bind FdUMP at this active site and its specific activity is proportionally lower. Thus, pure thymidylate synthase protein exists as a mixture of heterogeneous activity states. This condition is best examined by performing native PAGE on enzyme that has been incubated with excesses of FdUMP and CH2H4folate.

Chromatography, Affinity↗
PubMed1988

The removal of exogenous thiols from proteins by centrifugal column chromatography.

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.

Centrifugation↗
PubMed1987

Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes.

The true intracellular substrates for folate-dependent enzymes are folylpolyglutamates. We have used measurements of the Ki values of folylpolyglutamate dead end inhibitors to assess the relative affinities of folate-dependent enzymes for folate derivatives of different polyglutamate chain lengths. Studies of four enzymes from pig liver, methylenetetrahydrofolate reductase, serine hydroxymethyltransferase, methylenetetrahydrofolate dehydrogenase and thymidylate synthase, have indicated that folylpolyglutamate inhibitors are bound 3-500 fold more tightly than the corresponding monoglutamates. The individual enzymes differ in their selectivity for polyglutamate vs. monoglutamate inhibitors, and in the chain length associated with the greatest affinity of enzyme for inhibitor. We have also examined the effect of polyglutamate chain length on the catalytic parameters associated with folate substrates. Two enzymes, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase, show decreases in Km values for folylpolyglutamate substrates. Methylenetetrahydrofolate dehydrogenase shows no detectable differences in the catalytic parameters of polyglutamate vs. monoglutamate substrates and no change in the order of substrate addition or product release. Thymidylate synthase shows small effects of Km and Vmax values, but the order of addition of substrates and of release of products is reversed with polyglutamate as compared with monoglutamate substrates. Our studies with thymidylate synthase from L. casei have shown that the bacterial enzyme also exhibits a greatly increased affinity for polyglutamate vs. monoglutamate derivatives of folic acid, and that reversal in the order of substrate addition and product release also occurs with polyglutamate as compared with monoglutamate substrates. We have also studied the polyglutamate specificity of methionine synthase, which is responsible for the conversion of CH3-H4PteGlu1 into H4PteGlu1. This reaction is required for the incorporation of plasma folate into the cellular folate pool, because methyltetrahydrofolate is a poor substrate for folylpolyglutamate synthetase. Our studies demonstrate that CH3-H4PteGlu6, and suggest that incorporation of plasma CH3-H4PteGlu1 will only occur when methylenetetrahydrofolate reductase is inhibited by adenosylmethionine and cellular pools of CH3-H4PteGlu6 are at very low levels.

Animals↗
PubMed1986

Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei.

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.

Crystallography↗
PubMed1986

Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase.

The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗
PubMed1979

Modification of lysyl residues of dihydrofolate reductase with 2,4-pentanedione.

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) from an amethopterin-resistant strain of Lactobacillus casei was inactivated by 2,4-pentanedione. The inactivation appears to be due to the specific interaction of 2,4-pentanedione with lysyl residues. Inactivation is concomitant with with the modification of three lysyl residues. Both NADPH and dihydrofolate protect the enzyme against inactivation, suggesting that the critical residue(s) lies at or near their binding sites. Unlike native dihydrofolate reductase, 2,4-pentanedione-modified enzyme does not form binary complexes with either NADPH, dihydrofolate or amethopterin which are stable to gel filtration. Treatment of the modified enzyme with nucleophilic reagents such as hydroxylamine, failed to promote reactivation of the enzyme. Reactivation was achieved following gel filtration at pH 6.0 and was found to be dependent on the degree to which the enzyme was inactivated.

Arginine↗