KINETICS AND THERMODYNAMICS OF THE BINDING OF FORSKOLIN TO THE GALACTOSE-H-COLI( TRANSPORT PROTEIN, GALP, OF ESCHERICHIA)

Citation
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
Citations number
21
Categorie Soggetti
Biology
Journal title
ISSN journal
02646021
Volume
308
Year of publication
1995
Part
1
Pages
261 - 268
Database
ISI
SICI code
0264-6021(1995)308:<261:KATOTB>2.0.ZU;2-S
Abstract
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).