Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5g of enzyme is obtained from a 400 litre culture. The purification procedure, involving polyethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. ofapprox. 17900 and a turnover number of4s-(5OmM-triethanolamine/400mM-KCI, pH7.2, 25°C) with dihydrofolate and NADPH as substrates.The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.361uM-dihydrofolate; 0.78 uM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 x 106M-1; NADPH, >108M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.Dihydrofolate reductase (tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is responsible for maintaining the intracellular pool of tetrahydrofolate by reducing dihydrofolate (arising either by biosynthesis de novo or by the action of thymidylate synthetase on 5,10-methylenetetrahydrofolate) to tetrahydrofolate (Blakley, 1969). This enzyme is of considerable pharmacological interest as the site of action of a group of powerful chemotherapeutic agents, the 'anti-folates', which includes methotrexate, trimethoprim and pyrimethamine (Blakley, 1969;Baker, 1967).We are undertaking a detailed study of the binding of substrates, coenzymes and inhibitors to the dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei, principally by highresolution n.m.r. (nuclear-magnetic-resonance) spec-* Present address:

The structure of mouse L 1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0/~ resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 A resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X-ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty-one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.

By the use of molecular models of Escherichia coli dihydrofolate reductase (DHFR), analogues of trimethoprim (TMP) were designed which incorporated various 3'-carboxyalkoxy moieties in order to acquire ionic interactions with positively charged active-site residues. Certain of these compounds have shown exceptionally high affinity for this enzyme. For example, the 3'-(carboxypentyl)oxy analogue was found to be 55-fold more inhibitory than TMP toward E. coli DHFR (Ki = 0.024 nM vs. 1.32 nM for TMP). X-ray crystallographic studies of E. coli DHFR in binary complexes with TMP and two members of this acid-containing series of compounds defined the binding of these inhibitors and showed the carboxyl group of the latter two inhibitors to be ionically bound to Arg-57. These observations were in agreement with postulated binding modes that were based on receptor modeling.

The interactions of the coenzyme, NADPH, and of /7-aminobenzoyl-L-glutamate, a fragment of the substrate, with Lactobacillus casei dihydrofolate reductase have been studied by nuclear magnetic resonance spectroscopy. The aromatic proton resonances of p-aminobenzoyl-L-glutamate shift upfield in the presence of the enzyme by 0.41 and 0.58 ppm (protons ortho and meta to the glutamate moiety, respectively); the binding constant is 1.05 X 103 M~'. Similar chemical-shift changes on binding are observed with p-nitrobenzoyl-L-glutamate, but with p-aminobenzoyl-D-glutamate, no changes in chemical shift of the aromatic protons are observed, although it binds to the same site with a binding constant of 0.34 X 103 M~'. In the presence of NADPH, all three compounds bind approximately 3.5-fold more tightly, and the shifts of the aromatic protons of p-aminobenzoyl-and p-nitrobenzoyl-L-gluta-Dihydrofolate reductase(5,6,7,8-tetrahydrofolate:NADP oxidoreductase, EC 1.5.1.3) catalyzes the reduction of dihydrofolate to tetrahydrofolate. This reaction is of considerable importance in one-carbon metabolism, notably in thymidylate biosynthesis. Dihydrofolate reductases are also the target of a potent and important group of inhibitors of considerable chemotherapeutic interest (Blakley, 1969; Hitchings and Burchall, 1965). In order to improve our understanding of the mode of action of these folate antagonists at the molecular level, we are undertaking a detailed study of ligand binding to dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.We have begun by studying the binding of p-aminobenzoyl-L-glutamate (NHaBzGlu),* 1 a fragment of the substrate molecule, by high-resolution nmr spectroscopy. Both dihydrofolate and inhibitors such as methotrexate bind very tightly to dihydrofolate reductase (Kd ce. 10s-1010 M_l) and are thus in slow exchange on the nmr time scale. Fragments such as L-NH2BzG1u, however, bind much more weakly, are in rapid exchange, and can thus be studied much more conveniently. The low molecular weight of L. casei dihydrofolate reductase (17,500;Dann et al., 1974) has made it possible to study the nmr spectrum of the protein as well as the ligand in some detail.

The discovery of clonidine (1; Chart I) as a potent, centrally acting, hypotensive drug1-4 has led to detailed studies on the relationship between structure and hypotensive activity in the class of 2-(phenylimino)imidazolidines of the clonidine type. The majority of these investigations has considered variations of the substitution in the phenyl moiety of the molecules.

The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells has been studied by measuring the changes in chemical shift of nuclei of the ligand that accompanying binding. The 6- and 2',6'-proton chemical shifts of bound trimethoprim have been determined by transfer of saturation experiments, and the 2-carbon chemical shift has been determined by using [2-13C]trimethoprim. The changes in proton chemical shift are substantially smaller than those accompanying binding to bacterial dihydrofolate reductase [Cayley, P. J., Albrand, J. P., Feeney, J., Robert, G. C. K., Piper, E. A., & Burgen, A. S. V. (1979) Biochemistry 18, 3886]. It is shown that this difference arises largely from the fact that trimethoprim adopts different conformations when bound to mammalian and to bacterial dihydrofolate reductase. The proton chemical shifts are interpreted in terms of ring-current contributions from the two aromatic rings of trimethoprim itself and the nearby aromatic amino acid residues of the enzyme. The latter have been located by using the refined crystallographic coordinates of the Lactobacillus casei and Escherichia coli reductases in their complexes with methotrexate [Bolin, J. T., Filman, D. J., Matthews, D. A. & Kraut, J. (1982) J. Biol. Chem. 257, 13650], under the assumption that, as indicated by the 13C chemical shifts, the diaminopyrimidine ring of trimethoprim binds in the same way as does the corresponding part of methotrexate. With use of these assumptions, the conformation of trimethoprim bound to the dihydrofolate reductases from L. casei, E. coli, and L1210 cells has been calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

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