Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli

Journal Article · Tue Feb 26 00:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00222a023· OSTI ID:5745446

Falzone, C J; Benkovic, S J ^[1]; Wright, P E ^[2]

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1. Pennsylvania State Univ., University Park (United States)
2. Research Inst. of Scripps Clinic, La Jolla, CA (United States)

Two-dimensional {sup 1}H NMR methods and a knowledge of the X-ray crystal structure have been used to make resonance assignments for the amino acid side chains of dihydrofolate reductase from Escherichia coli complexed with methotrexate. The H7 proton on the pteridine ring of methotrexate was found to have NOEs to the methyl protons of Leu-28 which were assigned by using the L28F mutant. These NOEs indicated that the orientation of the methotrexate pteridine ring is similar in both solution and crystal structures. During the initial assignment process, it became evident that many of the resonances in this complex, unlike those of the folate complex, are severally broadened or doubled. The observation of two distinct sets of resonances in a ratio of approximately 2:1 was attributed to the presence of two protein isomers. Many of the side chains with clearly doubled resonances were located in the {beta}-sheet and the active site. Preliminary studies on the apoprotein also revealed doubled resonances in the absence of the inhibitor, indicating the existence of the protein isomers prior to methotrexate binding. In contrast to the methotrexate complex, the binary complex with folate and the ternary MTX-NADPH-DHFR complex presented a single enzyme form. These results are proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.

• OSTI ID: 5745446
• Journal Information: Biochemistry; (United States), Vol. 30:8; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
METHOTREXATE
ISOMERS
OXIDOREDUCTASES
NUCLEAR MAGNETIC RESONANCE
CRYSTALLOGRAPHY
ESCHERICHIA COLI
FOLIC ACID
LEUCINE
MOLECULAR STRUCTURE
OVERHAUSER EFFECT
PROTONS
X-RAY DIFFRACTION
AMINO ACIDS
ANTIMETABOLITES
AROMATICS
AZAARENES
BACTERIA
BARYONS
CARBOXYLIC ACIDS
COHERENT SCATTERING
DIFFRACTION
DRUGS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
MAGNETIC RESONANCE
MICROORGANISMS
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
PTERIDINES
RESONANCE
SCATTERING
VITAMIN B GROUP
VITAMINS
550201* - Biochemistry- Tracer Techniques