Quenching of thymidylate synthetase fluorescence by substrate analogs.

Quenching of fluorescence occurs when $\text{Lactobacillus casei}$ thymidylate synthetase is titrated with fluorodeoxyuridylate in the presence of 1-L-methylenetetrahydrofolate to form a ternary complex. Neither fluorodeoxyuridylate nor 1-L-methylenetetrahydrofolate added singly has any effect on enzyme fluorescence but d-L-methylenetetrahydrofolate alone causes quenching. Thus ternary complex formation and interaction with d-L-methylenetetrahydrofolate alter the environment of tryptophan residues in thymidylate synthetase in a similar manner.     

Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs.

Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).     

Quinazoline folate analogs inhibit the catalytic activity of thymidylate synthase but allow binding of 5-fluorodeoxyuridylate

We have investigated some unusual aspects of the inhibition of mammalian thymidylate synthase (TS) by the folate antimetabolite, 10-propargyl-5,8-dideaza-folic acid (CB 3717). From our results, we conclude that binding of CB 3717 metabolites to one subunit of L1210 TS modified the conformation of the second active site of this enzyme so that it retained the ability to bind 5-fluro-2'-deoxyuridine-5'-monophosphate (FdUMP) but not its catalytic activity. Exposure of intact mouse L1210 cells to CB 3717 resulted in inactivation of cellular TS activity, yet desalted cytosol preparations from these cells retained the ability to bind FdUMP. The same effect was found with several analogs of CB 3717. Complexes of FdUMP formed in vitro with TS from cells exposed to CB 3717 were covalent and co-migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with complexes of FdUMP, folate cofactor, and TS from cells not exposed to CB 3717. In the presence of dUMP, a tightly bound complex rapidly formed between isolated pure TS and the pentaglutamate of CB 3717 but not the monoglutamate form of this compound. Binding experiments using CB 3717 pentaglutamate-inhibited TS suggested a stoichiometry of 1 mol of FdUMP bound per mol of dimeric TS.     

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a ternary complex with enzyme/NAD+ similar to enzyme/NAD+/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.     

Comparative studies on thymidylate synthetase from P. berghei and mouse reticulocytes

Partially purified thymidylate synthetase from Plasmodium berghei and mouse reticulocytes was characterized. The mol. wt of the enzyme from P. berghei was about twice that from mouse reticulocytes. The optimum pH of the enzyme from P. berghei was found to be 6.5-7.5 while that from the host was 7.0-8.0. The enzyme from P. berghei was more susceptible to pH denaturation than the enzyme from reticulocytes. The enzyme from both sources differed in their Km values for substrates. The enzyme from reticulocytes was less sensitive to inhibition by substrate analogs than that from P. berghei.     

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

Electrophoretic identification of poly-γ-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes

A method has been developed to characterize the poly-γ-glutamates of 5,10-methyl-enetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy[³H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-γ-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates.     

The mechanism and structure of thymidylate synthetase

We have been involved in studies of the mechanism, inhibition and structure of the enzyme thymidylate synthetase. Knowledge of fundamental catalytic features of thymidylate synthetase has accumulated over the past decade, and will be described. Recently, we have been involved in studies of the x-ray crystallography of thymidylate synthetase, the first phase of which has been completed.     

X-ray studies on ternary complexes of maltodextrin phosphorylase

We report crystal structures of ternary complexes of maltodextrin phosphorylase with natural oligosaccharide and phosphate mimicking anions: nitrate, sulphate and vanadate. Electron density maps obtained from crystals grown in presence of Al(NO₃)₃ show a nitrate ion instead of the expected in the catalytic site. The trigonal is coplanar with the Arg569 guanidinium group and mimics three of the four oxygen atoms of phosphate. The ternary complex with sulphate shows a partial occupancy of the anionic site. The low affinity of the sulphate ion, observed when the α-glucosyl substrate is present in the catalytic channel, is ascribed to restricted space for the anion. Even lower occupancy is observed for the larger vanadate anion. The Malp/G5/ structure shows the partial occupancy of the oligosaccharide and the dislocation of the 380’s loop. This has been attributed to the formation of oligosaccharide vanadate derivatives (confirmed by capillary electrophoresis) that reduces their effective concentration. The difficulty to trap a ternary complex mimicking the ground state has been correlated to the apparent lower affinity that natural substrates show regarding the intermediates of the enzymatic reaction.     

Spectroscopic studies of pig kidney diamine oxidase-anion complexes

Complexes of pig kidney diamine oxidase with azide, thiocyanate, and cyanide have been characterized by EPR and circular dichroism spectroscopy. Cu(II) d-d bands are observed in the CD spectrum of the resting enzyme at ≈800 nm (12 500 cm⁻¹) and 575 nm (17 400 cm⁻¹). Anion binding produces characteristic changes in the CD spectra. N₃⁻/SCN⁻ → Cu(II) ligand-to-metal charge-transfer transitions are located at 390 nm (25 600 cm⁻¹) and 365 nm (27 400 cm⁻¹), respectively. In addition, an intense new band grew in at ≈500 nm (20 000 cm⁻¹) when azide or thiocyanate were added, which may be assigned as a Cu(II) electronic transition that gains rotational strength in the anion complex. EPR spectra established that the Cu(II)-anion complexes are tetragonal; however, the magnitudes of the anion-induced shifts in the EPR parameters were consistent with substantial perturbations of the Cu(II) electronic ground state in the thiocyanate and cyanide complexes. Prominent superhyperfine splitting was apparent in the EPR spectra of the diamine oxidase complexes with thiocyanate and cyanide. The superhyperfine structure is (at least) partially attributable to endogenous Cu(II) ligands, probably from imidazole.     

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.