Editing Dihydrofolate reductase (section)

=== General mechanism ===
[[File:DHFR Reaction Scheme.png|thumb|left|class=skin-invert-image|The reduction of dihydrofolate to tetrahydrofolate catalyzed by DHFR]]
DHFR catalyzes the transfer of a hydride from [[NADPH]] to [[dihydrofolate]] with an accompanying protonation to produce [[tetrahydrofolate]].<ref name="pmid15139807" /> 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.<ref name="pmid11502178" />

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.<ref name="Rod_2003" /><ref name="Wan_2014">{{cite journal | vauthors = Wan Q, Bennett BC, Wilson MA, Kovalevsky A, Langan P, Howell EE, Dealwis C | title = Toward resolving the catalytic mechanism of dihydrofolate reductase using neutron and ultrahigh-resolution X-ray crystallography | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 51 | pages = 18225–30 | date = December 2014 | pmid = 25453083 | pmc = 4280638 | doi = 10.1073/pnas.1415856111 | bibcode = 2014PNAS..11118225W | doi-access = free }}</ref> In a study using computational and experimental approaches, Liu ''et al'' conclude that the protonation step precedes the hydride transfer.<ref name="Liu_2014">{{cite journal | vauthors = Liu CT, Francis K, Layfield JP, Huang X, Hammes-Schiffer S, Kohen A, Benkovic SJ | title = ''Escherichia coli'' dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 51 | pages = 18231–6 | date = December 2014 | pmid = 25453098 | pmc = 4280594 | doi = 10.1073/pnas.1415940111 | bibcode = 2014PNAS..11118231L | doi-access = free }}</ref>

[[File:DHFR + NADPH + folate (Met20 loop).png|thumb|DHFR (Met20 loop highlighted) + NADPH + folate]]
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.<ref name="Liu_2014" /> 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.<ref name="Rod_2003" /><ref name="Czekster_2011">{{cite journal | vauthors = Czekster CM, Vandemeulebroucke A, Blanchard JS | title = Kinetic and chemical mechanism of the dihydrofolate reductase from ''Mycobacterium tuberculosis'' | journal = Biochemistry | volume = 50 | issue = 3 | pages = 367–75 | date = January 2011 | pmid = 21138249 | pmc = 3074011 | doi = 10.1021/bi1016843 }}</ref> 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. Asp27 plays 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.<ref name="Fierke_1987" /><ref name="Rod_2003" /><ref name="Czekster_2011" /> The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.<ref name="Wan_2014" /> A water molecule is proved to be involved in the protonation step.<ref name="Reyes_1995">{{cite journal | vauthors = Reyes VM, Sawaya MR, Brown KA, Kraut J | title = Isomorphous crystal structures of ''Escherichia coli'' dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications | journal = Biochemistry | volume = 34 | issue = 8 | pages = 2710–23 | date = February 1995 | pmid = 7873554 | doi = 10.1021/bi00008a039 }}</ref><ref name="Sawaya_1997" /><ref>{{cite journal | vauthors = Chen YQ, Kraut J, Blakley RL, Callender R | title = Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications | journal = Biochemistry | volume = 33 | issue = 23 | pages = 7021–6 | date = June 1994 | pmid = 8003467 | doi = 10.1021/bi00189a001 }}</ref> Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.<ref name="Shrimpton_2002">{{cite journal | vauthors = Shrimpton P, Allemann RK | title = Role of water in the catalytic cycle of E. coli dihydrofolate reductase | journal = Protein Science | volume = 11 | issue = 6 | pages = 1442–51 | date = June 2002 | pmid = 12021443 | pmc = 2373639 | doi = 10.1110/ps.5060102 }}</ref>