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250
1
200
0.8
150
0.6
100
0.4
cdk7/cyclin H
cdk2/cyclin A
cdc2/cyclin A
cdk2
cdk7
cdc2
50
0.2
0
0
0
0.5
1
1.5
2
2,5
0
0.2
0.4
0.6
0.8
1
1.2
cdks (µM)
cdk/cyclin (µM)
Figure 4.7 Binding of Mant-ATP to cyclin-dependent kinase (Cdk) and Cdk/cyclin com-
plex. Mant-ATP, the N-methyl-anthraniloyl moiety, is linked to 2
0
- and/or 3
0
-hydroxyl of
the ribose. The fluorescence of the Mant-group is excited at 350 nm and emission mon-
itored at 450 nm. The high sensitivity of the Mant-ATP was used to quantify the binding
of nucleotide to different Cdks in the free form (A) or in complex with their Cyclin part-
ner (B). Upon binding to Cdk and Cdk/cyclin complex, Mant fluorescence increased by
2.5- and 5-fold, respectively. In order to determine dissociation constant, the curves
were fitted using quadratic equation (Eq.
4.3
) (adapted from Morris et al.).
27
the binding of Mant-ATP to the cell cycle protein kinase cyclin-dependent
kinase (Cdk2) results in a 2.5-fold increase in fluorescence (
Fig. 4.7
) and
allows for the determination of the dissociation constant at the steady-state
level using either hyperbolic or quadratic equations, which take into
account the concentration of the enzyme and probe:
27,28
n
o
1
=
2
2
F
¼
F
ini
DF
ðÞ
ð
E
t
þ
L
þ
K
d
Þ
ð
E
t
þ
L
þ
K
d
Þ
4
E
t
L
=
2
E
t
½
4
:
3
where
F
is the observed relative fluorescence intensity,
F
ini
is the fluores-
cence intensity at the start of the titration,
DF
is the variation of the fluores-
cence intensity between the initial value and at a saturating concentration of
substrate,
E
t
is the total concentration of enzyme,
L
is the total concentration
of ligand, and
K
d
is the dissociation constant of the enzyme-ligand complex.
Bisubstrate nucleotides (Ap5A and Ap4A) have been designed to character-
ize enzymes harboring multiple nucleotide binding sites. An interesting
system was described to measure the affinities of nucleotide kinases
(NDP-, ADP-, AMP-, or TMP-kinases) for their substrates and inhibitors,
based on a fluorescent analog of the bisubstrate inhibitor diadenosine
pentaphosphate (AP5A):
a
,
g
-di[(3
0
-or2
0
-)-
O
-(
N
-methylanthraniloyl)
adenosine-5
0
] pentaphosphate (mAP5Am).
29-31
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