Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

Francis, Kevin; Stojković, Vanja; Kohen, Amnon

doi:10.1074/jbc.m113.507632

Cited by 39 publications

(77 citation statements)

References 46 publications

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“…However, as previously discussed and demonstrated, 23,24 single turnover rates do not exclusively represent C-H→C hydride transfer per se, but reflect several microscopic events including protein and ligand conformational changes that follow substrate binding (induced fit) and N5-H 2 F protonation that occur prior to the hydride transfer step. 54 The kinetic complexity resulting from the multi-step step nature of the single turnover measurement necessitates the use of intrinsic KIEs as carried out here to assess possible synergism between multiple residues of DHFR.…”

Section: Resultsmentioning

confidence: 85%

“…23,24,26,31–34 Hybrid quantum mechanical/molecular mechanics/molecular dynamics (QM/MM/MD) simulations have increasingly provided insight into the nature of conformational sampling in the DHFR-catalyzed reaction, predicting a network of coupled motions extending throughout the protein. 7,11,35–37 QM/MM/MD simulations and bioinformatics analysis (denoted as genomic coupling or co-evolution) 38,39 have suggested coupled motions in DHFR that correlates with its catalyzed chemistry.…”

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confidence: 99%

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Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis

2015

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Molecular dynamics calculations and bionformatic studies of dihydrofolate reductase (DHFR) have suggested a network of coupled motions across the whole protein that is correlated to the reaction coordinate. Experimental studies demonstrated that distal residues G121, M42 and F125 in E. coli DHFR participate in that network. The missing link in our understanding of DHFR catalysis is the lack of a mechanism by which such remote residues can affect the catalyzed chemistry at the active site. Here, we present a study of the temperature dependence of intrinsic kinetic isotope effects (KIEs) that indicates synergism between a remote residue in that dynamic network, G121, and the active site’s residue I14. The intrinsic KIEs for the I14A-G121V double mutant showed steeper temperature dependence (ΔEa(T-H)) than expected from comparison of the wild type and two single mutants. That effect was non-additive, i.e., ΔEa(T-H)G121V +ΔEa(T-H) I14A < ΔEa(T-H) double mutant, which indicates a synergism between the two residues. This finding links the remote residues in the network under investigation to the enzyme’s active site, providing a mechanism by which these residues can be coupled to the catalyzed chemistry. This experimental evidence validates calculations proposing that both remote and active site residues constitute a network of coupled promoting motions correlated to the bond activation step (C-H→C hydride transfer in this case). Additionally, the effect of I14A and G121V mutations on single turnover rates was additive rather than synergistic. Although single turnover rate measurements are more readily available and thus more popular than assessing intrinsic kinetic isotope effects, the current finding demonstrates that for these rates, which in DHFR reflect several microscopic rate constants, can fall short of revealing the nature of the C-H bond activation per se.

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Section: Resultsmentioning

confidence: 85%

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confidence: 99%

Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis

2015

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“…More detailed spectroscopic studies will be needed to elucidate the precise mechanistic consequences of these changes, but it is likely that mutation favorably alters the population distribution of ligand-bound conformations in both proteins, facilitating progression of the bound intermediates along the reaction coordinate (37). Both theoretical (45,46) and experimental (47)(48)(49) studies have shown that a dynamic network of coupled motions influences DHFR catalysis, and evidence suggests that this network has been maintained over billions of years of evolution (50,51).…”

Section: Discussionmentioning

confidence: 99%

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

Schulenburg¹,

Stark

Künzle

et al. 2015

Journal of Biological Chemistry

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Background: Intrinsic protein disorder has been hypothesized to be advantageous for protein evolution. Results: Parallel evolution of structured and disordered dihydrofolate reductases afforded comparable increases in activity without substantially changing the dynamic properties of the starting scaffolds. Conclusion: Ordered and disordered enzymes can be similarly evolvable. Significance: Understanding how structural (dis)order influences evolutionary pathways may aid efforts to engineer existing proteins for new tasks.

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“…4,9 Disruption of the optimized TRS in some ecDHFR mutants results in poor reorganization with broadly distributed DADs resulting in temperature-dependent KIEs. 10–15 …”

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confidence: 99%

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

et al. 2016

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Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general.

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Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

Cited by 39 publications

References 46 publications

Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis

Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis

Comparative Laboratory Evolution of Ordered and Disordered Enzymes

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

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