Biology Reference
In-Depth Information
of the (FixL)
2
(FixJ)
2
complex with ATP but affects neither ATP binding
(
K
d
¼
ca. 100
m
M) nor the (FixL)
2
(FixJ)
2
complex formation (
K
d
¼
ca.
4
m
M) (
Sousa, Gonzalez, & Gilles-Gonzalez, 2005
).
ADP produced from ATP bound in the kinase domain of
Sm
FixL acts as
an allosteric effector, reducing O
2
-binding affinity of the sensor domain in
Sm
FixL (
Nakamura et al., 2004
). The
K
d
(O
2
) values of the (FixL)
2
(FixJ)
2
complex are 56 and 218
m
M in the absence and presence of ADP, respec-
tively (
Nakamura et al., 2004
). O
2
-binding/dissociation kinetics measure-
ments by stopped flow method reveal that decrease in O
2
-binding affinity
is mainly caused by an increase of
k
off
(rate constant of O
2
dissociation)
(
Nakamura et al., 2004
).
Nakamura et al. (2004)
propose “a two-cylinder
reciprocating engine model” of the ADP-dependent acceleration of kinase
reaction, where ADP produced at the ATP-binding site in the FixL kinase
domain of one subunit in the (FixL)
2
(FixJ)
2
complex reduces the O
2
-
binding affinity of the other subunit of FixL in
trans
-acting manner.
4.1.1 O
2
regulates the activity of FixL
FixL contains a b-type haem and its kinase activity of FixL is regulated by O
2
binding to the haem (
Gilles-Gonzalez, Ditta, &Helinski, 1991
). Activities of
FixL can be evaluated by autophosphorylation of FixL and/or phosphoryl
group transfer to FixJ.
Tuckerman, Gonzalez, Dioum, and Gilles-
Gonzalez (2002)
report that O
2
binding to the haem in deoxy
Sm
FixL
(an external ligand-free Fe
2
þ
form) causes
>
100-fold decrease in the activity
of
Sm
FixJ phosphorylation (phosphoryl group transfer from
Sm
FixL to
Sm
FixJ), while autophosphorylation activity of FixL decreases by 15-fold.
They also report that the activity of
Sm
FixJ phosphorylation activity
decreases by 100-fold upon the oxidation of deoxy
Sm
FixL to met
Sm
FixL,
though met
Sm
FixL shows the same activity for autophosphorylation of
Sm
FixL as does deoxy
Sm
FixL (
Tuckerman et al., 2002
).
Akimoto,
Tanaka, Nakamura, Shiro, and Nakamura (2003)
, however, reported that
the met and deoxy forms of
Sm
FixL show comparable activities for both
autophosphorylation of
Sm
FixL and
Sm
FixJ phosphorylation. They demon-
strate that S
d
S bond formation at Cys301 causes aberrant kinase inactiva-
tion in met
Sm
FixL (
Akimoto et al., 2003
). The contents of the aberrant
inactive form with the S
d
S bond will vary in preparation to preparation.
Once the S
d
S bond is formed, it remains intact also in deoxy
Sm
FixL pro-
duced by reduction of met
Sm
FixL with sodium dithionite. On the other
hand, DTT treatment of met
Sm
FixL to produce deoxy
Sm
FixL cleaves
