Lactoperoxidase (
LPO
,
EC 1.11.1.7
) is a member of the mammalian heme peroxidase family which also includes thyroid peroxidase (
TPO
). These two enzymes have a sequence homology of 76%. The structure of
LPO
is known but not that of
TPO
. In order to determine the mode of binding of antithyroid drugs to thyroid peroxidase, we have determined the crystal structure of
LPO
complexed with an antithyroid drug, methimazole (
MMZ
) at 1.97 Å resolution.
LPO
was isolated from caprine colostrum, purified to homogeneity and crystallized with 20% poly(ethylene glycol)‐3350. Crystals of
LPO
were soaked in a reservoir solution containing
MMZ
. The structure determination showed the presence of two crystallographically independent molecules in the asymmetric unit. Both molecules contained one molecule of
MMZ
, but with different orientations.
MMZ
was held tightly between the heme moiety on one side and the hydrophobic parts of the side chains of Arg255, Glu258, and Leu262 on the opposite side. The back of the cleft contained the side chains of Gln105 and His109 which also interacted with
MMZ
. In both orientations,
MMZ
had identical buried areas and formed a similar number of interactions. It appears that the molecules of
MMZ
can enter the substrate‐binding channel of
LPO
in two opposite orientations. But once they reach the distal heme pocket, their orientations are frozen due to equally tight packing of
MMZ
in both orientations. This is a novel example of an inhibitor binding to an enzyme with two orientations at the same site with nearly equal occupancies.
Lactoperoxidase (LPO) belongs to mammalian heme peroxidase superfamily, which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO catalyzes the oxidation of a number of substrates including thiocyanate while TPO catalyzes the biosynthesis of thyroid hormones. LPO is also been shown to catalyze the biosynthesis of thyroid hormones indicating similar functional and structural properties. The binding studies showed that 2-mercaptoimidazole (MZY) bound to LPO with a dissociation constant of 0.63 µM. The inhibition studies showed that the value of IC was 17 µM. The crystal structure of the complex of LPO with MZY showed that MZY bound to LPO in the substrate-binding site on the distal heme side. MZY was oriented in the substrate-binding site in such a way that the sulfur atom is at a distance of 2.58 Å from the heme iron. Previously, a similar compound, 3-amino-1,2,4-triazole (amitrole) was also shown to bind to LPO in the substrate-binding site on the distal heme side. The amino nitrogen atom of amitrole occupied the same position as that of sulfur atom in the present structure indicating a similar mode of binding. Recently, the structure of the complex of LPO with a potent antithyroid drug, 1-methylimidazole-2-thiol (methimazole, MMZ) was also determined. It showed that MMZ bound to LPO in the substrate-binding site on the distal heme side with 2 orientations. The position of methyl group was same in the 2 orientations while the positions of sulfur atom differed indicating a higher preference for a methyl group.
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