Val Hs 143 with larger size hydrophobic residues, i.e., Ile Pf 263 and Ile Pf 272,
respectively. This results in the ineffectiveness of the larger molecules such as
brequinar and atovaquone to bind in PfDHODH. Hurt et al. in 2005 reported the
role of non-conserved residues in modifying the interactions of the inhibitor
(A77-1726) with the conserved amino acids. The replacement of Met Hs 43 and
Ala Hs 59 for Leu Pf 172 and Phe Pf 188, respectively, leads to alteration in the H-bond
pattern of the inhibitor with the conserved residues (His Pf 185, Arg Pf 265, and
Tyr Pf 528) at the end of the tunnel. Also, the replacement of Tyr Hs 147 for Cys Pf 276
leads to conformational changes in His Pf 185 resulting in changed interaction pattern
of the inhibitor for the two enzymes [62]. In summary, the non-conserved residues
present in the inhibitor binding site of the PfDHODH structure are Phe Pf 171
(Leu Hs 42), Met Pf 536 (Pro Hs 364), Leu Pf 172 (Met Hs 43), Phe Pf 188 (Ala Hs 59),
Leu Pf 176 (Gln Hs 47), Ile Pf 263 (Val Hs 134), Ile Pf 272 (Val Hs 134) [62, 75].
3.4 Inhibition of DHODH
L-DHO was observed to be the specific substrate of DHODH with a K m value of
5.2 ± 0.6 µM. D-DHO is not a substrate but inhibits the enzyme competitively
with K i of 1.4 mM concentration [48]. For the oxidation from DHO to orotate,
L-DHO diffuses passively from the cytosol to the intermembrane space of the
mitochondria where it binds tightly to the enzyme due to low K m value so that
enzyme shows maximum efficiency even at low concentrations of DHO. It was
observed that conversion of DHO to orotate is not the rate-limiting step, so
substrate-competitive inhibition will not be effective. This leaves the researchers
with two possibilities for inhibition of the enzyme, i.e., either by increasing the
intracellular accumulation of orotate or a lack of oxygen/its equivalent (inhibition of
electron receptor). However, intracellular accumulation of orotate inhibits dihydroorotase (enzyme catalyzing the formation of DHO from carbamylaspartate), thus
controlling the intracellular concentration of DHODH. Thus, the main center for
enzyme inhibition is obstructing the electron receptors [48].
Copeland et al. studied the role of N-terminal in enzyme inhibition in human
DHODH. It was observed that the essential catalytic region and site of inhibition are
located within 40 kDa area of truncated enzyme and the remaining 10 kDa of the
truncated N-terminal portion of the protein does not significantly disturb the catalytic action or inhibitor binding ability of the enzyme [76]. However, it was later
observed that the truncated enzyme only retains the activity under in vitro conditions and not under in vivo conditions [77]. This may be due to the removal of
signaling peptide and transmembrane helix which are responsible for cellular
localization and directing CoQ into the ubiquinone-binding tunnel.
Structure-Based Design of PfDHODH Inhibitors …
197
respectively. This results in the ineffectiveness of the larger molecules such as
brequinar and atovaquone to bind in PfDHODH. Hurt et al. in 2005 reported the
role of non-conserved residues in modifying the interactions of the inhibitor
(A77-1726) with the conserved amino acids. The replacement of Met Hs 43 and
Ala Hs 59 for Leu Pf 172 and Phe Pf 188, respectively, leads to alteration in the H-bond
pattern of the inhibitor with the conserved residues (His Pf 185, Arg Pf 265, and
Tyr Pf 528) at the end of the tunnel. Also, the replacement of Tyr Hs 147 for Cys Pf 276
leads to conformational changes in His Pf 185 resulting in changed interaction pattern
of the inhibitor for the two enzymes [62]. In summary, the non-conserved residues
present in the inhibitor binding site of the PfDHODH structure are Phe Pf 171
(Leu Hs 42), Met Pf 536 (Pro Hs 364), Leu Pf 172 (Met Hs 43), Phe Pf 188 (Ala Hs 59),
Leu Pf 176 (Gln Hs 47), Ile Pf 263 (Val Hs 134), Ile Pf 272 (Val Hs 134) [62, 75].
3.4 Inhibition of DHODH
L-DHO was observed to be the specific substrate of DHODH with a K m value of
5.2 ± 0.6 µM. D-DHO is not a substrate but inhibits the enzyme competitively
with K i of 1.4 mM concentration [48]. For the oxidation from DHO to orotate,
L-DHO diffuses passively from the cytosol to the intermembrane space of the
mitochondria where it binds tightly to the enzyme due to low K m value so that
enzyme shows maximum efficiency even at low concentrations of DHO. It was
observed that conversion of DHO to orotate is not the rate-limiting step, so
substrate-competitive inhibition will not be effective. This leaves the researchers
with two possibilities for inhibition of the enzyme, i.e., either by increasing the
intracellular accumulation of orotate or a lack of oxygen/its equivalent (inhibition of
electron receptor). However, intracellular accumulation of orotate inhibits dihydroorotase (enzyme catalyzing the formation of DHO from carbamylaspartate), thus
controlling the intracellular concentration of DHODH. Thus, the main center for
enzyme inhibition is obstructing the electron receptors [48].
Copeland et al. studied the role of N-terminal in enzyme inhibition in human
DHODH. It was observed that the essential catalytic region and site of inhibition are
located within 40 kDa area of truncated enzyme and the remaining 10 kDa of the
truncated N-terminal portion of the protein does not significantly disturb the catalytic action or inhibitor binding ability of the enzyme [76]. However, it was later
observed that the truncated enzyme only retains the activity under in vitro conditions and not under in vivo conditions [77]. This may be due to the removal of
signaling peptide and transmembrane helix which are responsible for cellular
localization and directing CoQ into the ubiquinone-binding tunnel.
Structure-Based Design of PfDHODH Inhibitors …
197