Proton nuclear magnetic resonance studies of the effects of ligand binding on ryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei
We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been asigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.
- Research Organization:
- National Inst. for Medical Research, London, England
- OSTI ID:
- 5589198
- Journal Information:
- Biochemistry; (United States), Vol. 19:11
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
OXIDOREDUCTASES
NMR SPECTRA
STRUCTURAL CHEMICAL ANALYSIS
COMPLEXES
DEUTERIUM
LACTOBACILLUS
LIGANDS
METABOLISM
NUCLEAR MAGNETIC RESONANCE
TRYPTOPHAN
AMINO ACIDS
AZOLES
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROGEN ISOTOPES
INDOLES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PYRROLES
RESONANCE
SPECTRA
STABLE ISOTOPES
550201* - Biochemistry- Tracer Techniques