Inhibitory Ternary Complex Research Articles

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 Å 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.

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)

Fluorine-19 nuclear magnetic resonance characterization of ternary complexes of folate derivatives, 5-fluorodeoxyuridylate and Lactobacillus casei thymidylate synthetase

Numerous biochemical techniques have been employed to characterize the covalent inhibitory ternary complex of thymidylate synthetase consisting of enzyme, 5-fluorodeoxyuridylate, and 5,10-methylenetetrahydrofolate. 19F NMR studies of this covalent ternary complex reveal a single, broad resonance centered at 12.7 ppm to higher shielding of free nucleotide, while the 5-fluorodexyuridylate-enzyme binary complex exhibits two resonances to higher shielding of free nucleotide, one at 1.4 ppm representing noncovalently bound ligand and the other at 34.5 ppm indicative of covalently bound 5,6-dihydro-5-fluorodeoxyuridylate. In order to follow the transformation of the latter binary complex to a ternary complex, we have employed 19F NMR to profile changes in the environment of the nucleotide which result from the interaction of folates with the coenzyme binding site. At low molar excesses of folates (5-fold), the effects observed in the 19F NMR spectrum fall into three major classes. (1) 5-Methyltetrahydrofolate exhibited a weak interaction with the binary complex. (2) Methotrexate and aminopterin, antifolate drugs, were observed to increase the exchange rate among the species detected in the 19F NMR spectrum of the binary complex. (3) Folate, dihydrofolate, and a series of tetrahydrofolate derivatives were found to shift the equilibrium of the binary complex toward the covalent 5,6-dihydro-5-fluorodeoxyuridylate-enzyme complex. With the latter folates the chemical shifts for the covalent species of these ternary complexes were found in the range of 35-40 ppm to higher shielding and are interpreted to reflect subtle differences in the strength and steric nature of the interaction of the folate ligand with the binary complex. These data illustrate that the latter folates promote the conversion of the enzyme-bound nucleotide to a species which would be poised to form the second covalent bond of the ternary complex, namely the linkage of the methylene group of the coenzyme with carbon 5 of the nucleotide.

Automated kinetic determination of lactate dehydrogenase isoenzymes in serum.

A steady-state kinetic method has been revised for measuring lactate dehydrogenase isoenzyme activities, which relates the inhibition of heart-type isoenzyme activity to the overall isoenzyme composition of the enzyme subunits. The method depends on the pH-dependent formation of an inhibitory ternary complex by the heart-type isoenzyme with NAD+ and pyruvate (if the reaction is measured by NADH oxidation). A preincubation step in the previous method is eliminated. The isoenzymes are measured by measuring the reduction of pyruvate in two different concentrations, which favor either the total or fractional activity, depending on the concentrations of pyruvate and the percentage of heart-type subunits. The method has been adapted to a centrifugal analyzer, which has speeded automated isoenzyme determinations, with an accuracy comparable to that for electrophoretic methods.