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

Abstract

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 aremore » proposed to reflect the ability of folate and NADPH to bind predominantly to one protein isomer.« less

Authors:
;  [1];  [2]
  1. Pennsylvania State Univ., University Park (United States)
  2. Research Inst. of Scripps Clinic, La Jolla, CA (United States)
Publication Date:
OSTI Identifier:
5745446
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 30:8; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
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

Citation Formats

Falzone, C J, Benkovic, S J, and Wright, P E. Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli. United States: N. p., 1991. Web. doi:10.1021/bi00222a023.
Falzone, C J, Benkovic, S J, & Wright, P E. Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli. United States. https://doi.org/10.1021/bi00222a023
Falzone, C J, Benkovic, S J, and Wright, P E. Tue . "Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli". United States. https://doi.org/10.1021/bi00222a023.
@article{osti_5745446,
title = {Evidence for two interconverting protein isomers in the methotrexate complex of dihydrofolate reductase from Escherichia coli},
author = {Falzone, C J and Benkovic, S J and Wright, P E},
abstractNote = {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.},
doi = {10.1021/bi00222a023},
url = {https://www.osti.gov/biblio/5745446}, journal = {Biochemistry; (United States)},
issn = {0006-2960},
number = ,
volume = 30:8,
place = {United States},
year = {1991},
month = {2}
}