COSOLVENT MODULATION OF THE TUBULIN-COLCHICINE CTPASE-ACTIVATING CONFORMATIONAL CHANGE - STRENGTH OF THE ENZYMATIC-ACTIVITY

The locus of action of cosolvent additives in the activation of the tu bulin-colchicine GTPase was investigated. The GDP off rates were slowe d down by the cosolvents in a manner that parallels their specific vis cosities, indicating that diffusion-controlled release of GDP may be r ate-limiting under the conditions of these studies. Yet, the net effec t of cosolvents was to increase the overall rate of GTP hydrolysis. Pr e-steady-state kinetics of liganded tubulin in the presence of 1%, w/v , poly(ethylene glycol) 6000 (PEG-6000) exhibited a burst of inorganic phosphate release indicating that the cosolvents act at an early step in the process. A similar conclusion was drawn from measurements of t he activation energy (E(a)) of the reaction, which showed that 3.4 M g lycerol decreased the value of E(a) to 10.6 kcal mol(-1) from 17.3 kca l mol(-1) in its absence. The observed difference in apparent binding free energies of the colchicine analogues allocolchicine (ALLO) and ,3 ,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC, or des-ring B c olchicine), when measured by fluorescence and enzyme activity titratio ns, identified the presence of a GTPase-activating protein conformatio nal transition subsequent to the physicochemical binding of the ligand s. The decrease of the apparent binding constant measured by enzyme ac tivity in dilute buffer relative to that measured by fluorescence [for ALLO, K-b(fluor) = 1.46 x 10(6) M(-1); K-b(enz act) = 1.1 X 10(5) M(- 1)] yielded the value of the enzyme-activating conformational transiti on constant, K-3 = 0.08. While neither 3.4 M glycerol nor 1%, w/v, PEG -6000 affected the apparent binding constant of ALLO measured by fluor escence, both increased that measured by enzyme titration. From the di fferences in the binding isotherms measured by enzymatic and fluoresce nce titrations, the fraction of active liganded tubulin was calculated to be 7.1, 26, and 79% in dilute buffer, 3.4 M glycerol, and 1%, w/v, PEG-6000, respectively. This led to strikingly similar values of the turnover number of the active form of the tubulin-drug complex, k(cat) (intrinsic) = (2.1 +/- 0.5) X 10(-3) s(-1), deduced from the kinetic m easurements under the different solution conditions. This value is 15 times greater than the k(cat)(apparent) measured in the absence of cos olvents [Perez-Ramirez, B., Shearwin, K. E., and Timasheff, S. N. (199 4) Biochemistry 33]. Thus, the intrinsic GTPase activity of the tubuli n-colchicine complex is, in fact, much stronger than that measured. Th e shifting of the equilibrium by the preferentially excluded cosolvent s from the inactive to the active species indicates the generation of a thermodynamically favorable environment for the active state of the protein by the cosolvents. In the case of 1%, w/v, PEG-6000, the favor able linkage free energy is -2.2 kcal mol(-1).