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

Zhang, Zhiquan; Rajagopalan, P. T. Ravi; Selzer, Tzvia; Benkovic, Stephen J.; Hammes, Gordon G.

doi:10.1073/pnas.0400091101

Cited by 71 publications

(86 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is due to environmental conditions that differ from those of a homogenous solvent, such as refractive index, local pH, ion concentrations, chemical groups and electric fields surrounding the probes. Dynamic quenching of the fluorophores 1,27 and static quenching that leads to dark states [29][30][31] can arise due to specific interactions between a dye and a macromolecule. In addition, the percentage of dye labeling is often incomplete.…”

Section: Introductionmentioning

confidence: 99%

Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

Majumdar¹,

Hickerson²,

Noller³

et al. 2005

Journal of Molecular Biology

View full text Add to dashboard Cite

Section: Introductionmentioning

confidence: 99%

Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

Majumdar¹,

Hickerson²,

Noller³

et al. 2005

Journal of Molecular Biology

View full text Add to dashboard Cite

“…An alternative path for the formation of EЈ⅐NADPH is the binding of E and NADPH to form E⅐NADPH, which then forms EЈ⅐NADPH. The second order rate constant for the overall combination of enzyme and NADPH is Ϸ10 7 M Ϫ1 ⅐s Ϫ1 and the overall equilibrium dissociation constant is Ϸ1 M (15,25).…”

Section: Resultsmentioning

confidence: 99%

“…Dihydrofolate reductase (DHFR) is an enzyme that has been extensively studied with many different methods and in many different laboratories (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). It catalyzes the reduction of dihydrofolate by nicotinamide adenine dinucleotide phosphate (NADPH) to tetrahydrofolate and NADP ϩ .…”

Section: Resultsmentioning

confidence: 99%

Conformational selection or induced fit: A flux description of reaction mechanism

Hammes

Chang

Oas

2009

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

Self Cite

506

642

View full text Add to dashboard Cite

The mechanism of ligand binding coupled to conformational changes in macromolecules has recently attracted considerable interest. The 2 limiting cases are the ''induced fit'' mechanism (binding first) or ''conformational selection'' (conformational change first). Described here are the criteria by which the sequence of events can be determined quantitatively. The relative importance of the 2 pathways is determined not by comparing rate constants (a common misconception) but instead by comparing the flux through each pathway. The simple rules for calculating flux in multistep mechanisms are described and then applied to 2 examples from the literature, neither of which has previously been analyzed using the concept of flux. The first example is the mechanism of conformational change in the binding of NADPH to dihydrofolate reductase. The second example is the mechanism of flavodoxin folding coupled to binding of its cofactor, flavin mononucleotide. In both cases, the mechanism switches from being dominated by the conformational selection pathway at low ligand concentration to induced fit at high ligand concentration. Over a wide range of conditions, a significant fraction of the flux occurs through both pathways. Such a mixed mechanism likely will be discovered for many cases of coupled conformational change and ligand binding when kinetic data are analyzed by using a fluxbased approach.kinetics ͉ binding ͉ folding ͉ coupled equilibria ͉ mechanism T he binding of ligands by macromolecules is crucial to a multitude of physiological processes. These include the cascade of reactions accompanying hormone binding to receptors (1, 2), enzyme reactions initiated by the binding of substrates to the enzyme (3, 4), gene regulation by the binding of molecules to DNA (5) and RNA (6), and the folding of unstructured proteins to produce biologically active molecules (7,8). In virtually all cases, the binding of ligands and conformational changes go hand in hand. Consequently, considerable effort has been expended in assessing the detailed mechanism by which ligand binding and conformational changes are coupled (3,4,9). Two limiting mechanisms are generally considered: (i) ''conformational selection,'' whereby the ligand selectively binds to a form of the macromolecule that is present only in small amounts, eventually converting the macromolecule to the ligand-bound conformation; and (ii) ''induced fit,'' (9) whereby ligand binds to the predominant free conformation followed by a conformational change in the macromolecule to give the preferred ligandbound conformation.The 2 limiting mechanisms can be written as:Conformational Selection: P weak º P tightInduced Fit: P weak ϩ L º P weak ⅐LIn these equations, P weak and P tight represent the tightly and weakly binding conformations of the protein (or other macromolecule), and L is the ligand. In point of fact, these 2 limiting mechanisms can be distinguished by kinetic measurements of the rate of the conformational change. In the simplest case, which often prevails, the ligand-bind...

show abstract

“…2), which are consistent with two parallel reaction pathways consisting of the ternary enzyme/ substrate (ES) Michaelis-Menten complex and the associated deprotonated form, ES −1 (separated by one ionization step). Although multiple ecDHFR ternary/binary complexes in solution have been reported (28), two catalytically competent ternary complexes, separated by protein ionization, were unknown (SI Appendix, Fig. S4).…”

mentioning

confidence: 99%

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

Liu

Francis

Layfield

et al. 2014

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

Self Cite

View full text Add to dashboard Cite

The reaction catalyzed by Escherichia coli dihydrofolate reductase (ecDHFR) has become a model for understanding enzyme catalysis, and yet several details of its mechanism are still unresolved. Specifically, the mechanism of the chemical step, the hydride transfer reaction, is not fully resolved. We found, unexpectedly, the presence of two reactive ternary complexes [enzyme:NADPH:7,8-dihydrofolate (E:NADPH:DHF)] separated by one ionization event. Furthermore, multiple kinetic isotope effect (KIE) studies revealed a stepwise mechanism in which protonation of the DHF precedes the hydride transfer from the nicotinamide cofactor (NADPH) for both reactive ternary complexes of the WT enzyme. This mechanism was supported by the pH-and temperature-independent intrinsic KIEs for the C-H→C hydride transfer between NADPH and the preprotonated DHF. Moreover, we showed that active site residues D27 and Y100 play a synergistic role in facilitating both the proton transfer and subsequent hydride transfer steps. Although D27 appears to have a greater effect on the overall rate of conversion of DHF to tetrahydrofolate, Y100 plays an important electrostatic role in modulating the pK a of the N5 of DHF to enable the preprotonation of DHF by an active site water molecule.kinetic isotope effect | mechanism | dihydrofolate reductase | synergism E scherichia coli dihydrofolate reductase (ecDHFR) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) through the transfer of a proR hydride from the C4 atom of NADPH to the C6 position of the dihydropterin ring of DHF (1). This enzyme is critical in maintaining the intercellular pool of DHF, which is subsequently used in the biosynthesis of purine nucleotides and some amino acids. Given its biological importance, DHFR is an important drug target (2, 3), and its function has been extensively studied (4-12). However, several fundamental mechanistic details remained incomplete. One unresolved issue is the chronological order of the DHF protonation and hydride transfer steps. Quantum mechanical/molecular mechanical (QM/MM) calculations favor a stepwise protonation-hydride transfer reaction mechanism (10, 13, 14), whereas recent experimental data were interpreted as a change in the reaction mechanism between a concerted process and a stepwise process as a function of pH (15).The nature of DHF N5 protonation is also not fully understood. D27 has been implicated in the protonation of the N5 position of NADPH (4,12,14,(16)(17)(18)) through a linked water molecule. However, it is not clear how the water molecule-promoted DHF protonation at the N5 of the pterin can change in response to protein conformation (19) and to different electrostatic environments. Recently, the Y100 residue, which is located only ca. 3.5 Å from both the amide of the nicotinamide and N8 of the pterin ring (Fig. 1), has been shown to play an important role in electrostatically facilitating the ecDHFR-catalyzed reaction (20, 21). The location of Y100 suggests that it may form hydrogen bonds with ...

show abstract

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

Cited by 71 publications

References 12 publications

Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

Measurements of Internal Distance Changes of the 30S Ribosome Using FRET with Multiple Donor–Acceptor Pairs: Quantitative Spectroscopic Methods

Conformational selection or induced fit: A flux description of reaction mechanism

Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100

Contact Info

Product

Resources

About