Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary A.; Benkovic, Stephen J.; Hammes, Gordon G.

doi:10.1073/pnas.172501499

Cited by 267 publications

(227 citation statements)

References 12 publications

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“…20; Fig. 1 A and B); these conformational fluctuations are consistent with single-molecule fluorescence results (19,21). Furthermore, MTX binds in an orientation in which the pterin ring is flipped 180°with respect to substrate, resulting in the MTX N1 atom being placed within H-bonding distance of the catalytic D27 residue (20,22).…”

supporting

confidence: 80%

“…1A). Also, in stopped-flow kinetics and single-molecule fluorescence experiments, MTX was shown to bind two different conformers of apo DHFR, and the enzyme displayed an additional conformational change attributed to opening and closing of the Met-20 loop (19). Interestingly, the x-ray structure of the DHFR⅐MTX complex reveals two molecules in the asymmetric unit in which the Met-20 loop adopts closed and occluded conformations (ref.…”

mentioning

confidence: 99%

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Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Bennett

Langan

Coates

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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supporting

confidence: 80%

mentioning

confidence: 99%

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Bennett

Langan

Coates

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…All single-molecule experiments were performed in triplicate, and, in all cases, well resolved fluorescent spots were observed. The DNA substrates were attached through a biotin-avidin interaction to the surface of a glass slide prepared with avidin on the surface (23). A solution containing 100 nM ssDNA or fDNA, 15 M BSA, and 0.1 M sodium phosphate (pH 7.0) was passed three times through the space between the coverslip and the avidin-coated slide.…”

mentioning

confidence: 99%

Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies

Zhang

Spiering

Trakselis

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Fig. 1. Protein-protein interactions between gp32 and gp59 on fDNA. (A) The fluorescence from individual molecules of fDNA with the proteins bound in the order as indicated at the side of each row. The gp32 protein is labeled with A488 (gp32 D ) and the gp59 protein is labeled with A555 (gp59 A ). The filter sets are described in Experimental Methods: F1 is for A488 emission, F2 for FRET between A488 and A555, and F3 for A555 emission. (B) Ensemble FRET studies of Oregon-green-488-maleimide-labeled gp59 titrated into a solution of 400 nM CPM-labeled gp32 and 100 nM fDNA. The fluorescence spectra of 400 nM CPM-gp32 alone (black line), the endpoint of the titration at 1 M Oregon-green-488-maleimide-gp59 (dark gray line), and several intermediate spectra (light gray lines) are shown. (C) Analysis of the donor quenching and acceptor sensitization plotted against the gp59 concentration determines the stoichiometry among gp32, gp59, and fDNA to be 1:1:1 with a calculated binding constant of Ϸ40 nM.

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“…The preparation and characterization of the fluorescent-labeled enzyme was as described (4). At pH 7.0, the steady-state turnover number of the fluorescent-labeled enzyme is 6.4 s Ϫ1 , Ϸ2.3-fold lower than the WT enzyme.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…The fluorescent dye Alexa 488 has been introduced into DHFR at the top of the structural loop by engineering a cysteine residue at position 18. The reaction of methotrexate with DHFR has been monitored by following fluorescence changes in Alexa 488 that accompany binding, and conformational changes of the enzyme have been characterized by stopped-flow and single-molecule fluorescence microscopy (4).…”

mentioning

confidence: 99%

Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate

Zhang

Rajagopalan

Selzer

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The interaction of dihydrofolate (H2F) and NADPH with a fluorescent derivative of H 2F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H 2 F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 ؋ 10 7 M ؊1 ⅐s ؊1 , and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H 2F with a bimolecular rate constant of 2.6 -5.7 ؋ 10 6 M ؊1 ⅐s ؊1 , with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s ؊1 at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme-substrate complexes exist. D ihydrofolate (H 2 F) reductase (DHFR) is a key enzyme for the biosynthesis of purines, thymidylate, and a number of amino acids. Its strategic location in metabolism has also made it a target for anticancer drugs. DHFR catalyzes the reaction of 7,8-H 2 F and NADPH to form 5,6,7,8-tetrahydrofolate. The mechanism of action of DHFR has been extensively probed to better understand the fundamental nature of enzyme catalysis. This is because of not only its physiological importance, but also its relatively easy purification and stability. These investigations include multiple structure determinations, steady-state and transient kinetic studies, theory, and single-molecule kinetics (cf. refs. 1-5).The structure of DHFR from Escherichia coli (molecular weight Ϸ18,000) is compact, with a loop that closes over the substrate binding sites. The mechanism of action of the enzyme involves multiple conformational changes that include domain rotation and interactions with structural loops (cf. refs. 1 and 2). Although the mechanism has been well characterized, it still remains an open question as to how the conformational changes that occur enhance the catalytic activity. Consequently, we have embarked on further investigation of the conformational coupling to catalysis by using transient and single-molecule kinetics.DHFR has a structural loop that closes over the substrates after they are bound. This is revealed by the x-ray structures and dynamic NMR measurements ...

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Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics

Cited by 267 publications

References 12 publications

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

Assembly of the bacteriophage T4 primosome: Single-molecule and ensemble studies

Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate

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