User:Bekidl/sandbox

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 Mechanism[edit]

The reduction of dihydrofolate to tetrahydrofolate catalyzed by DHFR.

General Mechanism[edit]

Since DHFR serves as an important model for mechanistic studies in enzymology, its catalytic mechanisms with different substrates have been investigated for a while using a wide range of methods including X-ray[1], NMR structures[2], molecular dynamics simulation[3], enzyme kinetic measurements[4][5], Raman spectroscopy analysis, and ensemble and single-molecule kinetics[6].

DHFR catalyzes the transfer of a hydride from NADPH to dihydrofolate with an accompanying protonation to produce tetrahydrofolate.[7] In the end, dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to NADP+. The high flexibility of Met20 and other loops near the active site play a role in promoting the release of the product, tetrahydrofolate. In particular the Met20 loop helps stabilize the nicotinamide ring of the NADPH to promote the transfer of the hydride from NADPH to dihydrofolate.[8]

EcDHFR in ternary complex with NADPH and the substrate

Conformational changes are critical in DHFR's catalytic mechanism.[4] The Met20 loop of DHFR is able to open, close or occlude the active site.[9][10] Correspondingly, three different conformations classified as the opened, closed and occluded states are assigned to Met20. In addition, an extra distorted conformation of Met20 was defined due to its indistinct characterization results.[10] The Met20 loop is observed in its occluded conformation in the three product ligating intermediates, where the nicotinamide ring is occluded from the active site. This conformational feature accounts for the fact that the substitution of NADP+ by NADPH is prior to product dissociation. Thus, the next round of reaction can occur upon the binding of substrate.[11]

Catalytic Kinetics in DHFR[edit]

The catalytic cycle of the reaction catalyzed by DHFR incorporates five important intermediate: holoenzyme (E:NADPH), Michaelis complex (E:NADPH:DHF), ternary product complex (E:NADP+:THF), tetrahydrofolate binary complex (E:THF), and THF‚NADPH complex (E:NADPH:THF). The kinetic studies showed that the product (THF) dissociation step from E:NADPH:THF to E:NADPH is the rate determining step during steady-state turnover.[11]

Studies on the kinetic mechanism of dehydrofolate reductase from Mycobacterium tuberculosis and Escherichia coli indicate that the mechanism of this enzyme is stepwise and steady-state random. Specifically, the catalytic reaction begins with the NADPH and the substrate attaching to the binding site of the enzyme, followed by the protonation and the hydride transfer from the cofactor NADPH to the substrate. However, two latter steps do not take place simultaneously in a same transition state.[9][12] In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer.[13]

Thus DHFR's enzymatic mechanism is shown to be pH dependent, particularly the hydride transfer step, since pH changes are shown to have remarkable influence on the electrostatics of the active site and the ionization state of its residues.[13] The acidity of the targeted nitrogen on the substrate is important in the binding of the substrate to the enzyme's binding site which is proved to be hydrophobic even though it has direct contact to water.[9][14] Asp27 is the only charged hydrophilic residue in the binding site, and neutralization of the charge on Asp27 may alter the pKa of the enzyme. Mutagenesis studies on important side chains show that Asp27 may play a critical role in the catalytic mechanism by helping with protonation of the substrate and restraining the substrate in the conformation favorable for the hydride transfer.[11][14] [9] The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.[12] In some other studies, a water molecule is proved to be involved in the protonation step[10][1][15]. Analysis of DHFR crystal structures as well as simulation studies have proved that the entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.[3]

R67 DHFR structure

R67 DHFR[edit]

Structure difference of substrate binding in E. coli and R67 DHFR

Due to its unique structure and catalytic features, R67 DHFR is widely studied. R67 DHFR is a type II R-plasmid-encoded DHFR without genetically and structurally relation to the E. coli chromosomal DHFR. It is a homotetramer that possesses the 222 symmetry with a single active site pore that is exposed to solvent.[16] This symmetry of active site results in the different binding mode of the enzyme: It can bind with two dihydrofolate (DHF) molecules with positive cooperativity or two NADPH molecules with negative cooperativity, or one substrate plus one, but only the latter one has the catalytical activity.[17] Compare with E. coli chromosomal DHFR, it has higher Km in binding dihydrofolate (DHF) and NADPH. The much lower catalytical kinetics show that hydride transfer is the rate determine step rather than product (THF) release.[18]

In the R67 DHFR structure, the homotetramer forms an active site pore. In the catalytical process, DHF and NADPH enters into the pore from opposite position. The π-π stacking interaction between NADPH's nicotinamide ring and DHF's pteridine ring tightly connect two reactants in the active site. However, the flexibility of p-aminobenzoylglutamate tail of DHF was observed upon binding which can promote the formation of the transition state.[19]

Respond to Peer Review[edit]

So the first thing we noticed, is that the first paragraph in the mechanisms section that you included on your sandbox but which you did not edit has several grammatical errors that could be corrected. As it stands it is difficult to read.

Reply: Thanks for your suggestions, we revised the text and make the correct for the sentences.

In regards to your contributions in sandbox, we noticed you do not include any information on coupled motions or distal residues potentially playing a role in catalysis (this is what we covered in class, so there are papers and a review on chalk that you could reference), so this is definitely something you could include.

Reply: In the revised version, we added more materials for the information of coupled motions and distal residues.

Also, maybe include authors by name in the article who made major contributions to the field/the information you are covering.

Reply: Thanks for your remind, we added the author's name in the revised version.

One minor thing we noticed is that toward the end of your second paragraph you ask a question. We think that you should rephrase this to not be a question, as you want to keep the information in your article encyclopedic and not in any way persuasive. Asking a question kind of leads the reader.

Reply: Good point! we rewrite the sentences and omit the question.

On a similar note, try to include your references earlier (after first sentence, as the modules told us to do), immediately when you bring up new information. Also, double-check your reference format. There is a way on sandbox to auto-cite where you just fill in a URL for the paper and it gives a very complete citation with links (in regular "edit," not "source edit") that you can use to directly access the paper. Right now your citations do not have that capability, and when the article goes live it is going to need to have this capability.

Reply: Thanks for your remind, we corrected the citation format in the new version.

Also we think that the enzyme complex formation described int he last sentence should be a quaternary complex not a ternary complex based upon the information mentioned previously referring to the enzyme as a homotetramer. Also, do you guys have any Pymol images that you want to include that could improve upon the one(s) on the article currently? This is something you could add that would be nice. Also your second paragraph is basically your only paragraph. Maybe you could separate it into two paragraphs instead so its easier to read.

Looking back, there are a few more points that could help. First, the beginning of the first R67 DHFR paragraph talks about the structure of the protein without giving any reference. Like we said earlier, a PyMol figure or two should make this clearer, but because the structure section is a bit bare bones, it might be worthwhile to expand on the 222 symmetry a bit and the structural difference between E. coli and R67 DHFR and to point out these differences in the PyMol figures.

Reply: Thanks for your suggestions. We tried to add a Pymol structure for the homotetramer, but we can only acquire monomer from PDB. The homotetramer structure proposed in the original article was a reconstruction figure by the author. Thus it is hard to regenerate a Pymol figure. However, based on your suggestion, a figure of its monomer structure made by our own and a homotetramer structure adopted from original paper are added to illustrate the structure information.

Starting in the second paragraph under R67, the D27 residue is discussed without much context about its role in catalysis in E. coli DHFR until later. It may be a good idea to discuss its significance earlier to streamline the article.

The information provided is really good, but the organization could use some improvement. Just a quick example, you may want to state the differences between E. coli and R67 DHFR and proceed to the experimental data acquired in separate paragraphs. This really does help make it more digestible for readers.

Reply: Thanks for your suggestion. A new table that summarize the difference between E. coli and R67 DHFR was made to make it more clear.

It may also help to put the names of the authors at the beginning of sentences, or at what aspect they are examining, and to discuss their work and findings/hypotheses. It's probably a good idea to go chronologically because then academic debates can be framed in this context and multiple sides can be given coverage.

Reply: Good point! in the revised version, we put the main contributor's name at the beginning of some sentences.

  1. ^ a b Reyes, Vicente M.; Sawaya, Michael R.; Brown, Katherine A.; Kraut, Joseph (1995-02-01). "Isomorphous Crystal Structures of Escherichia coli Dihydrofolate Reductase Complexed with Folate, 5-Deazafolate, and 5,10-Dideazatetrahydrofolate: Mechanistic Implications". Biochemistry. 34 (8): 2710–2723. doi:10.1021/bi00008a039. ISSN 0006-2960.
  2. ^ Falzone, Christopher J.; Wright, Peter E.; Benkovic, Stephen J. (1994-01-01). "Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis". Biochemistry. 33 (2): 439–442. doi:10.1021/bi00168a007. ISSN 0006-2960.
  3. ^ a b Shrimpton, Paul; Allemann, Rudolf K. (2002-06-01). "Role of water in the catalytic cycle of E. coli dihydrofolate reductase". Protein Science. 11 (6): 1442–1451. doi:10.1110/ps.5060102. ISSN 1469-896X. PMC 2373639. PMID 12021443.{{cite journal}}: CS1 maint: PMC format (link)
  4. ^ a b Antikainen, Nina M.; Smiley, R. Derike; Benkovic, Stephen J.; Hammes, Gordon G. (2005-12-01). "Conformation Coupled Enzyme Catalysis:  Single-Molecule and Transient Kinetics Investigation of Dihydrofolate Reductase". Biochemistry. 44 (51): 16835–16843. doi:10.1021/bi051378i. ISSN 0006-2960.
  5. ^ Maharaj, Geeta; Selinsky, Barry S.; Appleman, James R.; Perlman, Michael; London, Robert E.; Blakley, Raymond L. (1990-05-01). "Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase". Biochemistry. 29 (19): 4554–4560. doi:10.1021/bi00471a008. ISSN 0006-2960.
  6. ^ Rajagopalan, P. T. Ravi; Zhang, Zhiquan; McCourt, Lynn; Dwyer, Mary; Benkovic, Stephen J.; Hammes, Gordon G. (2002-10-15). "Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics". Proceedings of the National Academy of Sciences. 99 (21): 13481–13486. doi:10.1073/pnas.172501499. ISSN 0027-8424. PMC 129699. PMID 12359872.{{cite journal}}: CS1 maint: PMC format (link)
  7. ^ Schnell JR, Dyson HJ, Wright PE (2004). "Structure, dynamics, and catalytic function of dihydrofolate reductase". Annual Review of Biophysics and Biomolecular Structure. 33 (1): 119–40. doi:10.1146/annurev.biophys.33.110502.133613. PMID 15139807.
  8. ^ Osborne MJ, Schnell J, Benkovic SJ, Dyson HJ, Wright PE (August 2001). "Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism". Biochemistry. 40 (33): 9846–59. doi:10.1021/bi010621k. PMID 11502178.
  9. ^ a b c d Rod, Thomas H.; Brooks, Charles L. (2003-07-01). "How Dihydrofolate Reductase Facilitates Protonation of Dihydrofolate". Journal of the American Chemical Society. 125 (29): 8718–8719. doi:10.1021/ja035272r. ISSN 0002-7863.
  10. ^ a b c Sawaya, Michael R.; Kraut, Joseph (1997-01-01). "Loop and Subdomain Movements in the Mechanism of Escherichia coli Dihydrofolate Reductase:  Crystallographic Evidence,". Biochemistry. 36 (3): 586–603. doi:10.1021/bi962337c. ISSN 0006-2960.
  11. ^ a b c Fierke, Carol A.; Johnson, Kenneth A.; Benkovic, Stephen J. (1987-06-01). "Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli". Biochemistry. 26 (13): 4085–4092. doi:10.1021/bi00387a052. ISSN 0006-2960.
  12. ^ a b Wan, Qun; Bennett, Brad C.; Wilson, Mark A.; Kovalevsky, Andrey; Langan, Paul; Howell, Elizabeth E.; Dealwis, Chris (2014-12-23). "Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography". Proceedings of the National Academy of Sciences. 111 (51): 18225–18230. doi:10.1073/pnas.1415856111. ISSN 0027-8424. PMC 4280638. PMID 25453083.{{cite journal}}: CS1 maint: PMC format (link)
  13. ^ a b Liu, C. Tony; Francis, Kevin; Layfield, Joshua P.; Huang, Xinyi; Hammes-Schiffer, Sharon; Kohen, Amnon; Benkovic, Stephen J. (2014-12-23). "Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: Temporal order and the roles of Asp27 and Tyr100". Proceedings of the National Academy of Sciences. 111 (51): 18231–18236. doi:10.1073/pnas.1415940111. ISSN 0027-8424. PMC 4280594. PMID 25453098.{{cite journal}}: CS1 maint: PMC format (link)
  14. ^ a b Czekster, Clarissa M.; Vandemeulebroucke, An; Blanchard, John S. (2011-01-25). "Kinetic and Chemical Mechanism of the Dihydrofolate Reductase from Mycobacterium tuberculosis". Biochemistry. 50 (3): 367–375. doi:10.1021/bi1016843. ISSN 0006-2960. PMC 3074011. PMID 21138249.{{cite journal}}: CS1 maint: PMC format (link)
  15. ^ Chen, Y.-Q.; Kraut, Joseph; Blakley, Raymond L.; Callender, Robert (1994-06-01). "Determination by Raman Spectroscopy of the pKa of N5 of Dihydrofolate Bound to Dihydrofolate Reductase: Mechanistic Implications". Biochemistry. 33 (23): 7021–7026. doi:10.1021/bi00189a001. ISSN 0006-2960.
  16. ^ Narayana N, Matthews DA, Howell EE, Nguyen-huu X (November 1995). "A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site". Nature Structural Biology. 2 (11): 1018–25. PMID 7583655.
  17. ^ Bradrick TD, Beechem JM, Howell EE (September 1996). "Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein". Biochemistry. 35 (35): 11414–24. doi:10.1021/bi960205d. PMID 8784197.
  18. ^ Park H, Zhuang P, Nichols R, Howell EE (January 1997). "Mechanistic studies of R67 dihydrofolate reductase. Effects of pH and an H62C mutation". The Journal of Biological Chemistry. 272 (4): 2252–8. PMID 8999931.
  19. ^ Kamath G, Howell EE, Agarwal PK (October 2010). "The tail wagging the dog: insights into catalysis in R67 dihydrofolate reductase". Biochemistry. 49 (42): 9078–88. doi:10.1021/bi1007222. PMID 20795731.
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