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Title: sup 13 C and sup 15 N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase

Abstract

The state of protonation of substrates bound to mammalian dihydrofolate reductase (DHFR) has significance for the mechanism of catalysis. To investigate this, dihydrofolate and dihydropteroylpentaglutamate have been synthesized with {sup 15}N enrichment at N-5. {sup 15}N NMR studies have been performed on the binary complexes formed by bovine DHFR with these compounds and with (5-{sup 15}N)dihydrobiopterin. The results indicate that there is no protonation at N-5 in the binary complexes, and this was confirmed by {sup 13}C NMR studies with folate and dihydrofolate synthesized with {sup 13}C enrichment at C-6. The chemical shift displacements produced by complex formation are in the same direction as those which result from deprotonation of the N-3/C-4-O amide group and are consistent with at least partial loss of the proton from N-3. This would be possible if, as crystallographic data indicate, there is interaction of N-3 and the 2-amino group of the bound ligands with the carboxylate of the active site glutamate residue (Glu{sup 30}).

Authors:
; ;  [1];  [2];  [3];  [4]
  1. National Institute of Environmental Health Sciences, Research Triangle Park, NC (USA)
  2. Los Alamos National Laboratory, NM (USA)
  3. St. Jude Children's Research Hospital, Memphis, TN (USA)
  4. St. Jude Children's Research Hospital, Memphis, TN (USA) Univ. of Tennessee, Memphis (USA)
Publication Date:
OSTI Identifier:
6699646
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 29:5; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; FOLIC ACID; BIOSYNTHESIS; GLUTAMIC ACID; OXIDOREDUCTASES; NUCLEAR MAGNETIC RESONANCE; SUBSTRATES; IONIZATION; CARBON 13; CHEMICAL SHIFT; NITROGEN 15; AMINO ACIDS; AROMATICS; AZAARENES; CARBON ISOTOPES; CARBOXYLIC ACIDS; DRUGS; ENZYMES; EVEN-ODD NUCLEI; HEMATINICS; HEMATOLOGIC AGENTS; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; NITROGEN ISOTOPES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PTERIDINES; RESONANCE; STABLE ISOTOPES; SYNTHESIS; VITAMIN B GROUP; VITAMINS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Selinsky, B S, Perlman, M E, London, R E, Unkefer, C J, Mitchell, J, and Blakley, R L. sup 13 C and sup 15 N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase. United States: N. p., 1990. Web. doi:10.1021/bi00457a026.
Selinsky, B S, Perlman, M E, London, R E, Unkefer, C J, Mitchell, J, & Blakley, R L. sup 13 C and sup 15 N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase. United States. https://doi.org/10.1021/bi00457a026
Selinsky, B S, Perlman, M E, London, R E, Unkefer, C J, Mitchell, J, and Blakley, R L. Tue . "sup 13 C and sup 15 N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase". United States. https://doi.org/10.1021/bi00457a026.
@article{osti_6699646,
title = {sup 13 C and sup 15 N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase},
author = {Selinsky, B S and Perlman, M E and London, R E and Unkefer, C J and Mitchell, J and Blakley, R L},
abstractNote = {The state of protonation of substrates bound to mammalian dihydrofolate reductase (DHFR) has significance for the mechanism of catalysis. To investigate this, dihydrofolate and dihydropteroylpentaglutamate have been synthesized with {sup 15}N enrichment at N-5. {sup 15}N NMR studies have been performed on the binary complexes formed by bovine DHFR with these compounds and with (5-{sup 15}N)dihydrobiopterin. The results indicate that there is no protonation at N-5 in the binary complexes, and this was confirmed by {sup 13}C NMR studies with folate and dihydrofolate synthesized with {sup 13}C enrichment at C-6. The chemical shift displacements produced by complex formation are in the same direction as those which result from deprotonation of the N-3/C-4-O amide group and are consistent with at least partial loss of the proton from N-3. This would be possible if, as crystallographic data indicate, there is interaction of N-3 and the 2-amino group of the bound ligands with the carboxylate of the active site glutamate residue (Glu{sup 30}).},
doi = {10.1021/bi00457a026},
url = {https://www.osti.gov/biblio/6699646}, journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 29:5,
place = {United States},
year = {1990},
month = {2}
}