Nuclear magnetic resonance study of interaction of ligands with Streptococcus faecium dihydrofolate reductase labeled with (. gamma. -/sup 13/C)tryptophan

Journal Article · Fri Jan 01 04:00:00 EST 1982 · Biochemistry; (United States)

OSTI ID:6299731

London, R E ^[1]; Groff, J P; Cocco, L; Blakley, R L

---

1. Los Alamos National Lab., NM

Dihydrofolate reductase from Streptococcus faecium has been labeled with (..gamma..-/sup 13/C)tryptophan. We have determined changes occurring in the chemical shifts and line widths of the four resonances of the /sup 13/C NMR spectrum of the labeled enzyme, due to its interaction with various ligands. These include the coenzyme, NPDPH and related nucleotides, folate and its polyglutamate derivatives, and many inhibitors including methotrexate and trimethoprim. In addition, paramagnetic relaxation effects produced by a bound spin-labeled analogue of 2'-phosphoadenosine-5'-diphosphoribose on the tryptophan C/sup ..gamma../ carbons have been measured. Distances calculated from the relaxation data have been compared with corresponding distances in the crystallographic model of the NADPH-methotrexate ternary complex of Lactobacillus casei reductase. The paramagnetic relaxation data indicate that the two downfield resonances (1 and 2) correspond to tryptophans (W/sub A/ and W/sub B/) that are more remote from the catalytic site, and from the crystallographic model these are seen to be Trp-115 and Trp-160. The upfield resonances (3 and 4) that show broadening due to chemical exchange correspond to closer residues (W/sub C/ and W/sub D/), and these are identified with Trp-6 and Trp-22. However, the relaxation data do not permit specific assignments within the nearer and farther pairs. Although resonance 3, which is split due to chemical exchange, was formerly assigned to Trp-6, data obtained for the enzyme in the presence of various ligands are better interpreted if resonance 3 is assigned to Trp-22, which is located on a loop that joins elements of secondary structure and forms one side of the ligand-binding cavity.

• OSTI ID: 6299731
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 21; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AZOLES
BACTERIA
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
COENZYMES
ELECTRON SPIN RESONANCE
ENZYME INHIBITORS
ENZYMES
EVEN-ODD NUCLEI
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
INDOLES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGANDS
LIGHT NUCLEI
LINE WIDTHS
MAGNETIC RESONANCE
MICROORGANISMS
NADP
NMR SPECTRA
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PYRROLES
RESONANCE
SPECTRA
STABLE ISOTOPES
STREPTOCOCCUS
STRUCTURAL CHEMICAL ANALYSIS
TRACER TECHNIQUES
TRYPTOPHAN