Nuclear magnetic resonance studies on bacterial dihydrofolate reductase containing (methyl-/sup 13/C)methionine

Journal Article · Tue Jun 13 04:00:00 EDT 1978 · Biochemistry; (United States)

OSTI ID:6753672

Blakley, R L; Cocco, L; London, R E; Walker, T E; Matwiyoff, N A

(methyl-/sup 13/C)Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the /sup 13/C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T/sub 1/ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CH/sub 2/-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH/sub 2/-S bond is highly restricted so that the methyl group oscillates through a relatively narrow angular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably due to conformational transitions in the enzyme. However, NADP/sup +/ produced several changes,including movement of one resonance downfield by at least 1.7 ppM.

• Research Organization: Univ. of Iowa, Iowa City
• OSTI ID: 6753672
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 17:12; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
400103 -- Radiometric & Radiochemical Procedures-- (-1987)
550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANTIMETABOLITES
ANTIMITOTIC DRUGS
BACTERIA
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHEMICAL BONDS
COENZYMES
CONFIGURATION INTERACTION
DRUGS
ENZYMES
EVEN-ODD NUCLEI
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAGNETIC RESONANCE
METHIONINE
METHOTREXATE
MICROORGANISMS
NADP
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
RESONANCE
STABLE ISOTOPES
STREPTOCOCCUS
STRUCTURAL CHEMICAL ANALYSIS