%0 Journal Article
%A Eckenroth, Brian E.
%A Rould, Mark A.
%A Hondal, Robert J.
%A Everse, Stephen J.
%D 2007
%T Structural and Biochemical Studies Reveal Differences in the Catalytic Mechanisms
of Mammalian and Drosophila melanogaster Thioredoxin Reductases†
%U https://acs.figshare.com/articles/journal_contribution/Structural_and_Biochemical_Studies_Reveal_Differences_in_the_Catalytic_Mechanisms_of_Mammalian_and_i_Drosophila_melanogaster_i_Thioredoxin_Reductases_sup_sup_/3011479
%R 10.1021/bi602394p.s002
%2 https://acs.figshare.com/ndownloader/files/4712791
%K tetrapeptide binding pocket
%K enzyme
%K GR
%K DmTR
%K mTR 3
%K mouse mitochondrial TR
%K 2.4 Å resolution
%K protonating histidine residue
%K Cy
%K Sec
%K pyridine nucleotide disulfide oxidoreductases
%K Biochemical Studies Reveal Differences
%X Thioredoxin reductase (TR) from Drosophila melanogaster (DmTR) is a member of the
glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases and catalyzes the
reduction of the redox-active disulfide bond of thioredoxin. DmTR is notable for having high catalytic
activity without the presence of a selenocysteine (Sec) residue (which is essential for the mammalian
thioredoxin reductases). We report here the X-ray crystal structure of DmTR at 2.4 Å resolution (Rwork =
19.8%, Rfree = 24.7%) in which the enzyme was truncated to remove the C-terminal tripeptide sequence
Cys-Cys-Ser. We also demonstrate that tetrapeptides equivalent to the oxidized C-terminal active sites of
both mouse mitochondrial TR (mTR3) and DmTR are substrates for the truncated forms of both enzymes.
This truncated enzyme/peptide substrate system examines the kinetics of the ring-opening step that occurs
during the enzymatic cycle of TR. The ring-opening step is 300−500-fold slower when Sec is replaced
with Cys in mTR3 when using this system. Conversely, when Cys is replaced with Sec in DmTR, the
rate of ring opening is only moderately increased (5−36-fold). Structures of these tetrapeptides were
oriented in the active site of both enzymes using oxidized glutathione bound to GR as a template. DmTR
has a more open tetrapeptide binding pocket than the mouse enzyme and accommodates the peptide Ser-Cys-Cys-Ser(ox) in a cis conformation that allows for the protonation of the leaving-group Cys by His464‘,
which helps to explain why this TR can function without the need for Sec. In contrast, mTR3 shows a
narrower pocket. One possible result of this narrower interface is that the mammalian redox-active
tetrapeptide Gly-Cys-Sec-Gly may adopt a trans conformation for a better fit. This places the Sec residue
farther away from the protonating histidine residue, but the lower pKa of Sec in comparison to that of Cys
eliminates the need for Sec to be protonated.
%I ACS Publications