2. DIHYDROFOLATE REDUCTASE (DHFR)
• It is the reductase enzyme which catalyzes the
reduction of 7,8 Dihydrofolate through a
stereospecific hydride transfer from
NADPH(reduced form)cofactor to produced
5,6,7,8 Tetrahydrofolate product.
• It is found ubiquitously in all dividing cells of
prokaryotes and eukaryotes.
• MECHANISM OF ACTION:
• Tetrahydrofolate(FH4),which is produced from
the vitamin folate (B9) is primary one carbon
carrier in the body .
• While one carbons units are attached to FH4 at
N5 OR N10 Or form bridge between N5 and
N10 by donation of amino acids (majorly serine),
(histidine etc.) they can be oxidized or reduced
depends upon the requirement by biochemical
reaction .
3. • One of the major acceptor of this is deoxy-
uridine monophosphate which accepts
N5,N10 –methylene from FH4 to yield deoxy -
thymidine monophosphate(dTMP), purine
precursor.
• This is essential for purine and thymidylate
synthesis ,which are important for cell growth
and cell proliferation .
DHFR STRUCTURE:
• It is a cytosolic protein(186 amino
acids) of 21.3 kDa and uses NADPH
as cofactor.
• The DHFR sequence in humans
is 30% similar to that of E.coli and
70% similar to Other mammalian.
DHFR.
• The standard source of this enzyme is mammalian and
avian liver.
• In humans this enzyme is encoded by DHFR gene . It is
found in the q11-q22 region of chromosome 5.
• Four alpha helix connects successive beta strands.
• A central eight standard beta-pleated sheet makes up
the main feature of the polypeptide backbone folding of
DHFR.
.
4. • Seven of these strands are parallel and the eighth is antiparallel.
• The protein is divided into two subdomains , the adenosine
binding sub – domain and the loop domain .
• The adenosine binding sub -domain is larger of two and binds
adenoside moiety of NADPH .
• The loop subdomains consist of three loops, the Met-20 loop, the
F-G (phenylalanine –glycine) loop, and the G-H (glycine –
histidine)loop.
• Between these two loops there is active site .
• The active site is situated in N-terminal half of the sequence,which
includes a conserved Pro-trp dipeptide; the tryptophan has been
shown to be involved in the binding of substrate by the enzyme.
• The size of the active site is regulated by movements of the two
subdomains.
• E.COLI (ESCHERICHIA COLI) dihydrofolate reductase is an
important target of antibacterial medications .
• E.coli DHFR is small (18kb) protein with an α/ ꞵ structure consist
of a central eight stranded ꞵ -sheet and four flanking α-helices.
• The active site cleft of divides the protein into two structural
domains: 1).adenosine binding subdomain (light blue) and 2.)the
major subdomain : MET20(green),dark blue(F-G),and red (G-
H).
• The ternary complex of folate and NADP + illustrates the substrate
and cofactor binding site .
5. • There are five hydrogen bonds between folate
and DHFR active site :
1) Two H-bonds between ARG 70 and folate (
carboxylate group of glutamate )
2) Two H-bonds between GLU 30 and folate( NH2
and NH moiety of pyrimidine ring of pteridine ring)
• The GLU 30 is responsible for the protonation of
substrate and holds the substrate during the
catalytic activity of enzyme.
3) the other residues which interact with the folate
are:
1) One H- bond between ASN 64 and folate( C=O
of p-amino benzoic acid)
2) PHE 34 (through pi –pi interaction ) with folate (
phenyl ring of p-amino benzoic acid
FOLLOWING PICTURE
ILLUSTRATES HOW THE
COMPLEX OF DHFR AND
FOLATE FORM :
6. LIST OF VARIOUS INHIBITORS ACT ON THIS DHFR
ENZYME :
1. TRIMETHOPRIM –BLOCKS BACTERIAL
ENZYME
2. PYRIMETHAMINE ---PROTOZOAL
ENZYME
3. METHOTRAXATE ---MAMMALIAN
ENZYME
1.TRIMETHOPRIM : 5-(3,4,5-
TRIMETHOXYBENZYL) -2,4-PYRIMIDINE
-DIAMINE is a anti – bacterial drug used to treat
various infections like urinary tract infections,
respiratory tract infections and g.i.t. tract
infections.
• It is 50,000 TIMES more active against bacterial
DHFR enzyme than mammalian DHFR so, no
harm to human folate metabolism .
• It is used with sulphamethoxazole due to their
complementarity and synergistic action together
to achieve bactericidal action .
7. • 1.FIG. DEPICTING THE INTERACTION OF
TRIMETHOPRIM WITH THE DHFR AND
NADPH( DHRF OF LACTOBACILLUS CASEI)
• 2. FIG. : H-BOND
• INTERACTION B/W
• DHFR OF E.COLI
• AND TRIMETHOPRIM
• LIKE THE NATURAL SUBSTRATE , TRIMETHOPRIM
BINDS TO THE ENZYME ALONG THE NADPH IN A
TERNARY COMPLEX, IN WHICH THE REDUCING END
OF THE CO-FACTOR IS SITED NEAR TO PYRIMIDINE
RING OF TRIMETHOPRIM .
• TRIMETHOPRIM ,METHOTRAXATE ,PYRIMETHAMINE
HAVE 2,4-AMINOPYRIMIDINE RING WHICH
RESEMBLES THE PTERIDINE NUCLEUS OF
DIHYDROFOLATE.
• BY NMR AND X-RAY IT IS FOUND THAT N1 OF
TRIMETHOPRIM IS PROTONATED IN BOUND STATE .
• AND X-RAY CRYSTALLOGRAPHYALSO SHOWS THAT
TRIMETHOPRIM LOCATE IN A DEEP CLEFT OF THE
PROTEIN , WITH FAVOURABLE NON-COVALENT
INTERACTIONS HELPING TO MAINTAIN ITS POSSITION
.
• THIS BINDING MODE ENABLES IONIC INTERACTIONS
BETWEEN THE PROTONATED N1 AND CARBOXYLATE
GROUP OF THE CONSERVED AMINO ACID RESIDUE,
AND HYDROGEN BONDING INTERATIONS AS SHOWN
IN THE PICTURE.
• THESE COMPLEX INTERACTIONS HOLDING THE
TRIMETHOPRIM AND DHFR TOGETHER , RESULTS IN
POTENT INHIBITORY EFFECT OF TRIMETHOPRIM UPON
BACTERIAL DHFR.
8. METHOTRAXATE :
• IT is a dihydrofolate reductase
inhibitor that is used in the
treatment of autoimmune
inflammatory disease such as
rheumatid arithritis and also as a
antineoplastic drug .
10. Pyrimethamine :
• It is a directly acting inhibitor of plasdmodial
DHFRase.
• It gradually reduces the schizogony of
malarial parasite in blood .
• It is slowly acting erythrocytic schizontocide.
• STRUCTURE:
Moa:
Interactions between DHFR and Pyrimethamine:
