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

Journal Article · Tue Feb 06 00:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00457a026· OSTI ID:6699646

Selinsky, B S; Perlman, M E; London, R E ^[1]; Unkefer, C J ^[2]; Mitchell, J ^[3]; Blakley, R L ^[4]

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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)

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}).

• OSTI ID: 6699646
• Journal Information: Biochemistry; (USA), Vol. 29:5; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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