Enzyme

1.5.1.3 - Dihydrofolate reductase

Alternative Name(s)
  • Tetrahydrofolate dehydrogenase.

Catalytic Activity

(6S)-5,6,7,8-tetrahydrofolate + NADP(+) = 7,8-dihydrofolate + H(+) + NADPH

Cofactors

There are no Cofactors for this Enzyme

Reaction Mechanisms

    dihydrofolate reductase (bacterial) By Reference

    Dihydrofolate reductase catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) by stereospecific hydride transfer from a NADPH cofactor to the C6 atom of the pterin ring with concomitant protonation at N(5). DHFR plays a central role cell maintenance of THF reserves, which are essential for purine and thimidylate synthesis and hence for cell growth and proliferation. As DHFR is the sole source of THF, the enzyme is the Achilles heel of rapidly proliferating cells and, therefore, has been a major focus in the development of anticancer and antibacterial reagents.

    Much interest has been generated in this enzyme due to its potential as a target for antibacterial and anticancer drugs. It is ubiquitous throughout evolution, but always most important in cells which are dividing rapidly, hence its value as a drug target against infections and cancer.

    This mechanism proceeds in a single step in which the hydride ion is added to C6 with concomitant protonation of the N5 from a conserved water molecule. The active site residues are responsible for maintaining the steric placement of the substrates and Asp26, shown to be critical to the mechanism, is part of a hydrogen bonding network that modifies the pKa of the N5 atom from 2.4 to 6.5 when bound in a ternary complex. Further, and somewhat unusually, catalysis in this enzyme is dependent on a number of non-polar residues.
    Catalytic Residues
    AA Uniprot Uniprot Resid PDB PDB Resid
    Trp P00381 22 3dfr 21
    Asp P00381 27 3dfr 26
    Thr P00381 117 3dfr 116
    Leu P00381 95 3dfr 94
    Leu P00381 5 3dfr 4
    Leu P00381 20 3dfr 19
    Leu P00381 28 3dfr 27
    Phe P00381 31 3dfr 30
    Leu P00381 55 3dfr 54
    Step Components

    intermediate formation, overall reactant used, bimolecular nucleophilic addition, hydride transfer, proton transfer

    Step 1.

    The dihydrofolate substrate undergoes a tautomerisation reaction, resulting in the deprotonation of water.

    Products.

    The products of the reaction.

Reaction Parameters

  • Kinetic Parameters
    Organism KM Value [mM] Substrate Comment
    Haloarcula japonica 200 NADPH pH 8.0, 25°C
    Escherichia coli 547.3 NADPH pH 7.5, temperature not specified in the publication, cosubstrate: 10-formyl-dihydrofolate
    Moritella yayanosii 800 NADPH pH 7.0, 25°C, under atmospheric pressure
    Moritella japonica 1900 NADPH pH 7.0, 25°C, under atmospheric pressure
    Streptococcus pneumoniae 2000 NADPH wild-type, pH 7.0, 25°C
  • Temperature
    Organism Temperature Range Comment
    Moritella profunda 20 - 50 20°C: about 65% of maximal activity, 50°C: about 70% of maximal activity
  • pH
    Organism pH Range Comment
    Plasmodium falciparum 4 - 6.5 mutant W48Y, more than 60% of maximum activity
    Homo sapiens 4.2 - 5.6 half-maximal activities at pH 4.2 and pH 5.6
    Drosophila melanogaster 4.5 - 9
    Mycobacterium tuberculosis 5 - 9 activity at pH 9 is 8% of the activity at pH 5
    Enterococcus faecalis 5.3 - 8 half-maximal activities at pH 5.3 and pH 8.0

Associated Proteins

Protein name Organism
Viral dihydrofolate reductase SaHV-2
Dihydrofolate reductase type 15 Escherichia coli
Putative Dihydrofolate reductase HHV-8
Dihydrofolate reductase HdrA Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2)
Dihydrofolate reductase Human

Citations