James H. Freisheim

Functional arginine residues involved in coenzyme binding by dihydrofolate reductase.

Abstract Reaction of dihydrofolate reductase from amethopterin-resistant Lactobacillus casei with phenylglyoxal results in a complete loss of enzyme activity. This inactivation is concomitant with the modification of five of a total of eight arginine residues per mole of enzyme. In the presence of the reduced coenzyme, NADPH, two of the five reactive arginines are protected from chemical modification with complete retention of enzyme activity. The results suggest the involvement of essential arginine residues at or near the coenzyme binding site and thus at or near the active center of the enzyme.

An active center tryptophan residue in dihydrofolate reductase: chemical modification, sequence surrounding the critical residue, and structural homology considerations.

Abstract Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei contains three tryptophan residues and the amino acid sequence surrounding each tryptophan has been determined. Oxidation of one of these residues by N -bromosuccinimide at pH 6.5 can be correlated with the complete loss of enzymatic activity. Following denaturation in urea, the oxidized enzyme was alkylated with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Based on amino acid analyses and absorbance measurements at 410 nm, 2.2 mol of hydroxynitrobenzyl groups was incorporated per mol of protein. Presumably, hydroxynitrobenzyl adducts are formed with the two nonessential tryptophans. From the amino acid compositions of the two major thermolytic peptides containing the hydroxynitrobenzyl label and the partial sequences of two cyanogen bromide peptides containing the tryptophans, it was deduced that tryptophan-5 and tryptophan-129 were modified and, therefore, by difference, tryptophan-21 is the functional residue which becomes oxidized. The amino acid sequence surrounding tryptophan-21 is -Leu- -Trp-His-Leu-Pro-. In reductases from four other species, this region of the sequence is highly homologous; such a conservation in this vicinity of the primary structure may indicate a functional involvement. The proline residues at positions 20 and 24 may serve to position tryptophan-21 into the appropriate configuration for optimum substrate-binding interactions.

Circular dichroic titration of dihydrofolate reductase with TPNH

Abstract Dihydrofolate reductase from Streptococcus faecium shows a marked aromatic side chain Cotton effect in the 260–310 nm region of its circular dichroic spectrum. This effect consists of three distinct ellipticity bands with maxima centered at 305 nm, 295 nm and 270 nm. Titration of the enzyme with TPNH to a 1:1 stoichiometry results in the generation of an extrinsic Cotton effect at ca. 340 nm and a decrease in the magnitude of the side chain Cotton effect. This is the first such example of a TPNH-generated extrinsic Cotton effect. The data suggest the involvement of tryptophyl residues in coenzyme binding.

High-performance liquid chromatography of methotrexate analogs containing terminal lysine or ornithine and their dansyl derivatives

A procedure utilizing a reverse-phase semipreparative high-performance liquid chromatography column and a binary solvent system consisting of trifluoroacetic acid and 1-propanol has been developed for the semipreparative scale purification and analytical identification of four newly synthesized analogs of methotrexate. The methotrexate analogs containing a lysine or an ornithine residue in place of a terminal glutamate residue together with their respective dansyl derivatives were purified in milligram quantities by the procedures described.

Dihydrofolate reductase and thymidylate synthetase in strains of Streptococcus faecium resistant to pyrimethamine, chlorguanide triazine, trimethoprim, and amethopterin☆

Abstract Strains of Streptococcus faecium highly resistant to pyrimethamine, chlorguanide triazine or amethopterin exhibited increased levels of dihydrofolate reductase and thymidylate synthetase which varied from 2- to 40-fold. The increases in synthetase were always greater than the corresponding increases in reductase. A strain of the same organism resistant to trimethoprim, however, showed no increases in either enzyme. Studies on the partially purified reductases indicate that the pyrimethamine-resistant enzyme differs strikingly in certain molecular properties. This reductase had a pH optimum at about 5.0 whereas the other four enzymes showed optimum activity at about pH 6.O. It also showed altered inhibition profiles requiring from 4 times as much pyrimethamine to 40 times as much chlorguanide triazine as the sensitive enzyme to obtain 50% inhibition.