Biology Reference
In-Depth Information
state),
51,52
which is an important metabolic variable,
influencing many aspects of cell function like growth,
apoptosis, and reductive biosynthesis. In addition, by
redox signaling, they control the activation of a number
of transcription factors and hence regulate a broad range
of cellular functions.
53
e
55
The two redox systems physi-
ologically play many roles in different organisms and on
the other hand are also pathophysiological factors for a
variety of human diseases including cancer, viral
disease, Alzheimer's disease, and others
56
e
59
and hence
serve as vital drug targets for cancer therapy and other
disease treatments.
60
e
64
Although the thioredoxin
system and glutaredoxin system share a number of func-
tions, they are not just simple duplicate systems; TRX
and GRX act on different substrates.
65,66
TRX but not
GRX, for example, has been implicated in the reduction
of APE1.
67
e
70
The thiol/disulfide exchange reaction involves recog-
nition and reduction of an oxidized protein including
a disulfide bond by a reduced redox factor such as
TRX or GRX/GSH with a Cys in the redox factor acting
as a nucleophile.
48,49
Thioredoxins (TRXs) comprise
a large family of structurally conserved proteins that
serve as general protein disulfide oxidoreductases along
with thioredoxin reductase and NADPH
48,71
and can
reduce disulfide bonds in a variety of proteins.
72
TRXs
all include an active site motif Cys-X-X-Cys within
a common structural motif, the TRX fold,
52,54,73
a four-
stranded
b
-sheet surrounded by three
a
-helices
(
Figure 11.5
). The active site motif is located on the
loop connecting
b
-sheet 1 and
a
-helix 1. The N-terminal
Cys residue in the active site is surface exposed and has
a low pKa value; for example, Cys 32 in human TRX has
an estimated pKa of 6.3,
74
while the C- terminal Cys is
buried in the molecule and has a much higher pKa
value. The low pKa value of the N-terminal Cys has
been proposed to arise from the partial positive charge
from the dipole moment associated with alpha-helix 2
75
or alternatively may be due to its hydrogen bond to
the C-terminal Cys.
76
The nucleophilicity of the thio-
late group of the Cys is increased by the low pKa.
The proposed reaction mechanism of disulfide reduc-
tion by thioredoxin is as follows: the N-terminal
cysteine thiolate of TRX acts as a nucleophile and
attacks the target disulfide resulting in a transient
mixed disulfide intermediate, which is in turn reduced
by the C-terminal active site Cys residue generating
a dithiol in the target protein and a disulfide in thiore-
doxin.
52,73,77
This disulfide is reduced by electron
transfer from FADH
2
, and the resulting FAD is then
reduced by electron transfer from NADPH.
48
The
mechanism by which TR reduces TRX back to the
dithiol involves the formation of a selenylsulfide in
the active site of TR
48
as shown in
Figure 11.4
.
A second redox active site located in the other subunit
of the dimeric TR contains two thiols that reduce the
selenylsulfide back to a thiol and selenol with the
resultant formation of a disulfide bond.
The glutaredoxin system differs from the thioredoxin
system in the use of glutathione (glutamyl-cysteinyl-
glycine, GSH) as a cofactor but also uses NADPH,
a flavoprotein, glutathione reductase, and glutare-
doxin.
65,66,71
This system also works through a cascade
of disulfide oxidation and reduction. Glutaredoxins
(GRXs) are small redox enzymes of approximately 100
amino-acid residues that are structurally very similar
to thioredoxins retaining the same fold and active sites.
However, there is some variation in the actives sites of
GRXs, which include Cys-X-X-Cys or Cys-X-X-Ser.
GRXs catalyze the reversible reduction of substrate
protein disulfides resulting in oxidation of the GRXs
through a mechanism similar to that for TRXs. Oxidized
GRXs are reduced non-enzymatically by glutathione,
and then the oxidized glutathionine disulfide (GSSG)
is reduced by glutathione reductase at the expense of
NADPH.
66
Peroxiredoxins are yet another group of redox
proteins; they are responsible for sensing hydrogen
peroxide in the cell and serve as catalysts to detoxify
this extremely reactive molecule.
78
These enzymes
lack the C-X-X-C motif found in thioredoxins or glutare-
doxins but still require two Cys residues for activity.
79
The redox active Cys residues are located in different
subunits of the dimeric structure approximately 9
˚
from one another.
79
Prior to a local unfolding event
near the dimer interface, the nucleophilic thiolate is
sequestered and presumably protected from oxidation.
The resolving thiolate is located near the C-terminus of
the molecule and upon local unfolding is placed in close
proximity to the nucleophilic thiolate.
79
Overoxidation
of peroxiredoxins to the sulfenic or sulfinic acid state
can occur as a result of reduction of hydrogen
peroxide
78
and must be repaired by sulfiredoxin.
80,81
FIGURE 11.5
The structures of reduced and oxidized thioredoxin
are shown as ribbon renderings (gray) with redox active Cys residues,
black stick representations. The redox active Cys residues are Cys 32
and Cys 35. Formation of a disulfide bond in thioredoxin minimally
perturbs the structure of the protein.
