Kinetics of substrate, coenzyme, and inhibitor binding to Escherichia coli dihydrofolate reductase

Cayley, P. Jane; Dunn, Susan M. J.; King, Roy W.

doi:10.1021/bi00507a034

Cited by 110 publications

(144 citation statements)

References 11 publications

Supporting

Mentioning

134

Contrasting

Order By: Relevance

“…Thus this mechanistic possibility is consistent with all of the data. It should be noted, however, that the rate constants for the interconversion of E and EЈ for the WT enzyme have been estimated to be about 3 ϫ 10 Ϫ2 s Ϫ1 , much smaller than observed in the single-molecule studies (3). The binding of methotrexate to the WT enzyme also involves binding to both E and EЈ (3).…”

Section: Discussionmentioning

confidence: 83%

See 1 more Smart Citation

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

Rajagopalan

Zhang

McCourt

et al. 2002

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

271

216

View full text Add to dashboard Cite

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s ؊1 and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.enzyme mechanisms ͉ protein dynamics ͉ fluorescence microscopy

show abstract

Section: Discussionmentioning

confidence: 83%

“…Because of its important metabolic role, DHFR has been a target for anti-cancer drugs. One of these drugs is methotrexate that has a binding dissociation constant in the nanomolar region (3).…”

mentioning

confidence: 99%

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

Rajagopalan

Zhang

McCourt

et al. 2002

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

271

216

View full text Add to dashboard Cite

show abstract

“…However, as previously mentioned, the existence of multiple conformers of the free enzyme seems quite probable. The slow interconversion of multiple conformers of the free enzyme has been observed (7,11,12).…”

Section: Discussionmentioning

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.

View full text Add to dashboard Cite

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 ...

show abstract

“…Dihydrofolate reductase (DHFR) is a relevant exception. In the absence of substrate and cofactor, it partitions into two native states (N1 or E1 and N2 or E2) (Cayley et al 1981). These two states are in slow exchange on the NMR chemical shift timescale and give rise to two sets of resonances across the whole protein (Falzone et al 1991).…”

Section: Structural Effectsmentioning

confidence: 99%

Loop anchor modification causes the population of an alternative native state in an SH3‐like domain

Knappenberger

Lecomte

2007

Protein Science

View full text Add to dashboard Cite

Many stably folded proteins are proposed to contain long, unstructured loops. A series of hybrid proteins (EbE1-4) containing the folded scaffold of photosystem I accessory protein E (PsaE), an SH3-like protein, and the 40-residue heme-binding loop of cytochrome b 5 was created to inspect the dependence of thermodynamic and kinetic parameters on the residues at the interface of folded and flexible regions. Compared to the simplest hybrid (EbE1), the chimeras differed by Gly insertions (EbE2, EbE3) or an asymmetric four-residue restructuring of loop termini (EbE4). NMR spectroscopy indicated that the chimeras retained the PsaE topology; native and unfolded state solubilities, however, were affected to varying degrees. Thermal and chemical denaturation experiments revealed that the EbE2 and EbE1 constructs resulted in a modest destabilization of the PsaE core, whereas apparent stability was increased by >5 kJ/mol in EbE4. EbE3 aggregated at mM concentrations and was not studied in detail. EbE4 populated two native states (N1 and N2), which differed by hydrophobic core packing and C-terminal interactions. At room temperature, the population ratio (;3-4:1) favored the state whose spectroscopic properties most resembled those of PsaE (N1). EbE4 also demonstrated altered folding kinetics, displaying multiple slow phases related to the population of intermediates and possibly N2. It was concluded that loop anchors can affect protein properties, including stability, via short-range effects on local structure and long-range communication with the packed hydrophobic core. Modification of the attachment points appears to be a possible stepping stone in the transition from one three-dimensional structure to another.

show abstract

Kinetics of substrate, coenzyme, and inhibitor binding to Escherichia coli dihydrofolate reductase

Cited by 110 publications

References 11 publications

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

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

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

Loop anchor modification causes the population of an alternative native state in an SH3‐like domain

Contact Info

Product

Resources

About