User:Nhattrongdo/sandbox

An Error has occurred retrieving Wikidata item for infobox Dihydrofolate reductase, or DHFR, is an

Function

Dihydrofolate reductase converts

Found in all organisms, DHFR has a critical role in regulating the amount of tetrahydrofolate in the cell. Tetrahydrofolate and its derivatives are essential for

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, NMR structures, molecular dynamics simulation, enzyme kinetic measurements, Raman spectroscopy analysis, and ensemble and single-molecule kinetics.^{[13][14][15][16][17] [18]}

DHFR catalyzes the transfer of a hydride from

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.[19]

Conformational changes are critical in DHFR's catalytic mechanism.^[13] The Met20 loop of DHFR is able to open, close or occlude the active site.^[20][21] 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.^[21] 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.^[19]

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.^[20][22] In a study using computational and experimental approaches, Liu et al conclude that the protonation step precedes the hydride transfer.^[23]

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.^[23] 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.^[20][24] 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.^{[19][24] [20]} The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.^[22] In some other studies, a water molecule is proved to be involved in the protonation step^[21][17][25]. 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.^[15]

Clinical significance

Dihydrofolate reductase deficiency has been linked to megaloblastic anemia.^[9] Treatment is with reduced forms of folic acid. Because tetrahydrofolate, the product of this reaction, is the active form of folate in humans, inhibition of DHFR can cause functional folate deficiency. DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor synthesis. Trimethoprim, an antibiotic, inhibits bacterial DHFR while methotrexate, a chemotherapy agent, inhibits mammalian DHFR. However, resistance has developed against some drugs, as a result of mutational changes in DHFR itself.^[26]

DHFR mutations cause a rare autosomal recessive inborn error of folate metabolism that results in megaloblastic anemia, pancytopenia and severe cerebral folate deficiency which can be corrected by folinic acid supplementation .^[27]

Therapeutic applications

Since folate is needed by rapidly dividing cells to make thymine, this effect may be used to therapeutic advantage.

DHFR can be targeted in the treatment of cancer. DHFR is responsible for the levels of tetrahydrofolate in a cell, and the inhibition of DHFR can limit the growth and proliferation of cells that are characteristic of cancer.

Bacteria also need DHFR to grow and multiply and hence inhibitors selective for bacterial DHFR have found application as antibacterial agents.[30]

Classes of small-molecules employed as inhibitors of dihydrofolate reductase include diaminoquinazoline & diaminopyrroloquinazoline,^[36] diaminopyrimidine, diaminopteridine and diaminotriazines.^[37]

Potential anthrax treatment

Dihydrofolate reductase from Bacillus anthracis (BaDHFR) a validated drug target in the treatment of the infectious disease, anthrax. BaDHFR is less sensitive to trimethoprim analogs than is dihydrofolate reductase from other species such as Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. A structural alignment of dihydrofolate reductase from all four species shows that only BaDHFR has the combination phenylalanine and tyrosine in positions 96 and 102, respectively.

BaDHFR's resistance to trimethoprim analogs is due to these two residues (F96 and Y102), which also confer improved kinetics and catalytic efficiency.^[38] Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.^[38]

As a research tool

DHFR has been used as a tool to detect

Dihydrofolate reductase has been shown to interact with GroEL^[39] and Mdm2.^[40]

Interactive pathway map

Click on genes, proteins and metabolites below to link to respective articles.^{[§ 1]}

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|alt=Fluorouracil (5-FU) Activity edit]]

Fluorouracil (5-FU) Activity edit

1. ^ The interactive pathway map can be edited at WikiPathways: "FluoropyrimidineActivity_WP1601".

References