Skip to main content
SpringerLink
Log in

1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase

  • Published:
European Biophysics Journal Aims and scope Submit manuscript

Abstract

The binding of trimethoprim and [1,3,2-amino-15N3]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by 15N and 1H NMR spectroscopy. 15N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The 15N chemical shifts and 1H-15N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme.

The N1-proton resonance in the complex has been assigned using the 15N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160±10s-1 at 313 K, with an activation energy of 75 (±9) kJ·mole-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bachovchin WW, Roberts JD (1978) Nitrogen-15 nuclear magnetic resonance spectroscopy. The state of histidine in the catalytic triad of α-lytic protease. Implications for the charge-relay mechanism of peptide-bond cleavage by serine proteases. J Am Chem Soc 100:8041–8047

    Google Scholar 

  • Baker BR (1967) Design of active-site-directed irreversible enzyme inhibitors. John Wiley, New York

    Google Scholar 

  • Baker DJ, Beddell CR, Champness JN, Goodford PJ, Norrington FEA, Smith DR, Stammers DK (1981) The binding of trimethoprim to bacterial dihydrofolate reductase. FEBS Lett 126:49–52

    Google Scholar 

  • Baker DJ, Beddell CR, Champness JN, Goodford PJ, Norrington FEA, Roth B, Stammers DK (1983) X-ray studies of the binding of trimethoprim, methotrexate, pyrimethamine and two trimethoprim analogues to bacterial dihydrofolate reductase. In: Blair JD (ed) Chemistry and biology of pteridines. W de Gruyter, Berlin, pp 545–549

    Google Scholar 

  • Birdsall B, Burgen ASV, Roberts GCK (1980) Binding of coenzyme analogues to Lactobacillus casei dihydrofolate reductase: binary and ternary complexes. Biochemistry 19:3723–3731

    Google Scholar 

  • Birdsall B, Bevan AW, Pascual C, Roberts GCK, Feeney J, Gronenborn A, Clore GM (1984) Multinuclear NMR characterisation of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex. Biochemistry 23:4733–4742

    Google Scholar 

  • Blakley RL, Cocco L, Montgomery JA, Temple C, Jr., Roth B, Daluge S, London RE (1983) Catalytic site of dihydrofolate reductase. In: Blair JA (ed) Chemistry and biology of pteridines. W de Gruyter, Berlin, pp 215–221

    Google Scholar 

  • Bolin JT, Filman DJ, Matthews DA, Kraut J (1982) Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 Å resolution. I. General features and binding of methotrexate. J Biol Chem 257:13650–13662

    Google Scholar 

  • Brown DJ, Teitei T (1965) Pyrimidine reactions, part VII. Methylation of substituted 2,4-diaminopyrimidines. J Chem Soc 755-761

  • Cayley PJ, Albrand JP, Feeney J, Roberts GCK, Piper EA, Burgen ASV (1979) Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase. Biochemistry 18:3886–3895

    Google Scholar 

  • Clore GM, Kimber BJ, Gronenborn AM (1983) The 1-1 hard pulse: a simple and effective method of water resonance suppression in FT 1H NMR. J Magn Reson 54:170–173

    Google Scholar 

  • Cocco L, Groff JP, Temple C, Jr., Montgomery JA, London RE, Matwiyoff NA, Blakley RL (1981) 13C nuclear magnetic resonance study of protonation of methotrexate and aminopterin bound to dihydrofolate reductase. Biochemistry 20:3972–3978

    Google Scholar 

  • Cocco L, Roth B, Temple C, Jr., Montgomery JA, London RE, Blakley RL (1983) Protonated state of methotrexate, trimethoprim and pyrimethamine bound to dihydrofolate reductase. Arch Biochem Biophys 226:567–577

    Google Scholar 

  • Cresswell RM, Mentha JW (1975) US patent 3878252

  • Cresswell RM, Mentha JW, Seaman RJ (1972) US patent 3697512

  • Dann JG, Ostler G, Bjur RA, King RW, Scudder P, Turner PC, Roberts GCL, Burgen ASV, Harding NGL (1976) Large scale purification and characterisation of dihydrofolate reductase from a methotrexate resistant strain of Lactobacillus casei. Biochem J 157:559–571

    Google Scholar 

  • Doddrell DM, Pegg DT, Bendall MR (1982) Distortionless enhancement of NMR signals by polarization transfer. J Magn Reson 48:323–327

    Google Scholar 

  • Duthaler RO, Roberts JD (1978) Effects of solvent, protonation and N-alkylation on the 15N chemical shifts of pyridine and related compounds J Am Chem Soc 100:4969–4973

    Google Scholar 

  • Englander W, Kallenbach N (1984) Hydrogen exchange and structural dynamics of proteins and nucleic acids. Qu Rev Biophys 16:521–655

    Google Scholar 

  • Erickson JS, Mathews CK (1972) Spectral changes associated with binding of folate compounds to bacteriophage T4 dihydrofolate reductase. J Biol Chem 247:5661–5667

    Google Scholar 

  • Franken HD, Rüterjans H, Müller F (1984) Nuclear magnetic resonance investigation of 15N-labelled flavins, free and bound to Megasphaera elsdenii apoflavodoxin. Eur J Biochem 138:481–489

    Google Scholar 

  • Gronenborn A, Birdsall B, Hyde E, Roberts GCK, Feeney J, Burgen ASV (1981) 1H and 31P NMR characterisation of two conformations of the trimethoprim-NADP+-dihydrofolate reductase complex. Mol Pharmacol 20:145–153

    Google Scholar 

  • Gueron M, Leroy JL, Griffey RH (1983) Proton nuclear magnetic relaxation of 15N-labeled nucleic acids via dipolar coupling and chemical shift anisotropy. J Am Chem Soc 105:7262–7266

    Google Scholar 

  • Hood K, Roberts GCK (1978) Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase. Biochem J 171:357–366

    Google Scholar 

  • Hore PJ (1983) Solvent suppression in Fourier transform nuclear magnetic resonance. J Magn Reson 55:283–300

    Google Scholar 

  • Kanamori K, Roberts JD (1983a) 15N nuclear magnetic resonance study of benzenesulfonamide and cyanate binding to carbonic anhydrase. Biochemistry 22:2658–2664

    Google Scholar 

  • Kanamori K, Roberts JD (1983b) 15N NMR studies of biological systems. Acc Chem Res 16:35–41

    Google Scholar 

  • Kearns DR (1984) NMR studies of conformational states and dynamics of DNA. CRC Crit Rev Biochem 15:237–290

    Google Scholar 

  • Levy GC, Lichter RL (1979) Nitrogen-15 nuclear magnetic resonance spectroscopy. John Wiley, New York

    Google Scholar 

  • Martin GJ, Martin ML, Gouesnard JP (1981) 15N NMR spectroscopy. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams MN, Hoogsteen K (1977) Dihydrofolate reductase: X-ray structure of the binary complex with methotrexate. Science 197:452–455

    Google Scholar 

  • Matthews DA, Bolin JT, Filman DJ, Volz KW, Kraut J (1983) Dihydrofolate reductase: the stereochemistry of inhibitor selectivity. In: Blair JA (ed) Chemistry and biology of pteridines. W de Gruyter, Berlin, pp 435–443

    Google Scholar 

  • Morris GA (1980) Sensitivity enhancement in 15N NMR: polarization transfer using the INEPT pulse sequence. J Am Chem Soc 102:428–429

    Google Scholar 

  • Morris GA, Freeman R (1979) Enhancement of nuclear magnetic resonance signals by polarization transfer. J Am Chem Soc 101:760–762

    Google Scholar 

  • Poe M, Greenfield NJ, Hirshfield JM, Hoogsteen K (1974) Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli: binding of several folates and pteridines as minitored by ultraviolet difference spectroscopy. Cancer Biochem Biophys 1:7–11

    Google Scholar 

  • Poe M, Bennett CD, Donoghue D, Hirshfield JM, Williams MN, Hoogsteen K (1975) Mammalian dihydrofolate reductase; porcine liver enzyme. In: Pfleiderer W (ed) Chemistry and biology of pteridines. W de Gruyter, Berlin, pp 51–58

    Google Scholar 

  • Reid BR (1981) NMR studies on RNA structure and dynamics. Annu Rev Biochem 50:969–996

    Google Scholar 

  • Roberts GCK (1983) The interaction of substrates and inhibitors with dihydrofolate reductase. In: Blair JA (ed) Chemistry and biology of pteridines. W de Gruyter, Berlin, pp 197–214

    Google Scholar 

  • Roberts GCK, Feeney J, Burgen ASV, Daluge S (1981) The charge state of trimethoprim bound to Lactobacillus casei dihydrofolate reductase. FEBS Lett 131:65–88

    Google Scholar 

  • Roth B, Strelitz JZ (1969) The protonation of 2,4-diaminopyrimidines. I. Dissociation constants and substituent effects. J Org Chem 34:821–836

    Google Scholar 

  • Roth K, Kimber BJ, Feeney J (1980) Data shift accumulation and alternate delay accumulation techniques for overcoming the dynamic range problem. J Magn Reson 41:302–309

    Google Scholar 

  • Rüterjans H, Kaun E, Hull WE, Limbach HH (1982) Evidence for tautomerism in nucleic acid base pairs. 1H NMR study of 15N-labelled tRNA. Nucl Acid Res 10:7027–7039

    Google Scholar 

  • Schimmel PR, Redfield AG (1981) Transfer RNA in solution: selected topics. Annu Rev Biophys Bioeng 9:181–221

    Google Scholar 

  • Schuster II, Roberts JD (1979) 15N nuclear magnetic resonance spectroscopy. Effects of hydrogen bonding and protonation on nitrogen chemical shifts in imidazoles. J Org Chem 44:3864–3867

    Google Scholar 

  • Städeli W, von Philipsborn W, Wick A, Kompis I (1980) 15N NMR. Studies of aminopyridines, aminopyrimidines and of some diazine N-oxides. Helv Chim Acta 63:504–522

    Google Scholar 

  • Volz KW, Matthews DA, Alden RA, Freer ST, Hansch C, Kaufman BT, Kraut J (1982) Crystal structure of avian dihydrofolate reductase containing phenyltriazine and NADPH. J Biol Chem 257:2528–2536

    Google Scholar 

  • Witanowski M, Stefaniak L, Webb GA (1981) Nitrogen NMR Spectroscopy. Annu Rep NMR Spectrosc 11B:1–486

    Google Scholar 

Download references

Author information

Author notes

  1. A. W. Bevan (Recipient of an SERC CASE Studentship)

    Present address: Burroughs Wellcome Co., Research Triangle Park, North Carolina, USA

Authors and Affiliations

  1. Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, NW7 1AA, London, UK

    A. W. Bevan (Recipient of an SERC CASE Studentship), G. C. K. Roberts & J. Feeney

  2. Burroughs Wellcome Co., Research Triangle Park, North Carolina, USA

    L. Kuyper

Authors
  1. A. W. Bevan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. C. K. Roberts
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Feeney
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. Kuyper
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bevan, A.W., Roberts, G.C.K., Feeney, J. et al. 1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase. Eur Biophys J 11, 211–218 (1985). https://doi.org/10.1007/BF00261997

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00261997

Key words

Search

Navigation