Gem. Martin et al., KINETICS AND THERMODYNAMICS OF THE BINDING OF FORSKOLIN TO THE GALACTOSE-H-COLI( TRANSPORT PROTEIN, GALP, OF ESCHERICHIA), Biochemical journal, 308, 1995, pp. 261-268
The binding of the transport inhibitor, forskolin, to the galactose-H symporter, GalP, of Escherichia coli was evaluated by equilibrium and
time-resolved fluorescence measurements. A quench in protein fluoresc
ence of 8-12% was observed upon the binding of forskolin. The overall
dissociation constant (K-d) for forskolin determined by fluorescence t
itration ranged between 1.2 and 2.2 mu M, which is similar to that rep
orted from equilibrium dialysis measurements of the binding of [H-3]fo
rskolin (K-d = 0.9-1.4 mu M). The kinetics of forskolin binding were m
easured by stopped-flow fluorescence methods, The protein fluorescence
was quenched in a biphasic manner; the faster of these two rates was
dependent on the concentration of forskolin and was interpreted as the
initial binding step from which both the association (k(on)) and diss
ociation (k(off)) rate constants were determined. The association and
dissociation rate constants were 5.3-6.2 mu M(-1).s(-1) and 5.1-11.5 s
(-1) respectively, and the K-d was calculated to be 1.5 mu M. The bind
ing of forskolin was inhibited by D-galactose, but not by L-galactose,
and displacement by sugar provided an additional method to calculate
the dissociation rate constant for forskolin (k(off) = 12.4-13.0 s(-1)
). The rate of the slow change in protein fluorescence (3-5 s(-1)) was
independent of the forskolin concentration, indicating an isomerizati
on of the transporter between different conformations, possibly outwar
d- and inward-facing forms. These kinetic parameters were determined a
t a series of temperatures, so that the thermodynamics of forskolin bi
nding and transporter re-orientation could be analysed. The binding pr
ocess was entropically driven (Delta S = 83.7 J . K-1 . mol(-1); Delta
H = 8.25 kJ . mol(-1)), similar to that for cytochalasin B, which is
also an inhibitor of GalP. Measurements of the binding of [H-3]forskol
in by equilibrium dialysis revealed competitive displacement of bound
forskolin by cytochalasin B, possibly suggesting that the sugar, forsk
olin and cytochalasin B binding sites are overlapping; the K(d)s for f
orskolin and cytochalasin B were calculated to be 0.85 mu M and 4.77 m
u M respectively, and the concentration of binding sites was 10.2 nmol
. mg(-1).