Methotrexate, aminopterin, and folate have been synthesized with 90% enrichment of C-2 with 13C. 13C nuclear magnetic resonance has been used to examine the state of protonation of the pteridine ring of these compounds under various conditions and gives much more clear-cut results than most other methods. For the free compounds the following pK values were obtained: methotrexate, 5.73 +/- 0.02 (N-1); aminopterin, 5.70 +/- 0.03 (N-1); folic acid, 2.40 (N-1) and 8.25 +/- 0.05 (N-3, O-4 amide group). The state of protonation of these compounds when complexed to dihydrofolate reductase (isoenzyme 2 from Streptococcus faecium) was also studied over the pH range 6--10. The resonance from bound methotrexate showed a constant chemical shift over the whole pH range studied, and it is inferred that in the complex the pteridine ring remains protonated to at least pH 10. The same result was obtained for the binary complex of aminopterin with the reductase and for either methotrexate or aminopterin in ternary complex with reductase and NADPH4. The latter is an inhibitor of the reductase competitive with NADPH. However, folate bound to the reductase in either the binary or the ternary complex shows the same protonation behavior as in the free state. The data indicate that the association constant for binding of methotrexate is increased enough when protonation of N-1 occurs to account for the enhanced binding of methotrexate as compared with folate.

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Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the l3C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T\ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CHi-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH2-S bond is highly restricted so that the methyl group oscillates through a relatively narrowangular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably 4 he structure of dihydrofolate reductase is currently under intensive study in a number of laboratories. Interest in this enzyme derives in part from its clinical relevance. The reductase is the target of methotrexate, a drug used in the treatment of various types of cancer, and bacterial reductase is strongly and specifically inhibited by trimethoprim which is now used extensively in combination with sulfa drugs for treating a variety of infectious diseases.Although studies of the amino acid sequence, the effects of chemical modification, and x-ray diffraction of the crystalline protein have made important contributions to understanding the structure of the reductase, NMR studies are able to make a unique contribution. The method does not perturb the structure of the enzyme and is potentially capable of giving information about the internal motions of amino acid residues and about the interactions of residues at the active site with a great variety of substrates and inhibitors in binary or ternary f From the