Cosolvent modulation of the tubulin-colchicine GTPase-activating conformational change: strength of the enzymatic activity.
The locus of action of cosolvent additives in the activation of the tubulin-colchicine GTPase was investigated. The GDP off rates were slowed down by the cosolvents in a manner that parallels their specific viscosities, indicating that diffusion-controlled release of GDP may be rate-limiting under the conditions of these studies. Yet, the net effect of cosolvents was to increase the overall rate of GTP hydrolysis. Pre-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 the activation energy (Ea) of the reaction, which showed that 3.4 M glycerol decreased the value of Ea to 10.6 kcal mol-1 from 17.3 kcal mol-1 in its absence. The observed difference in apparent binding free energies of the colchicine analogues allo-colchicine (ALLO) and 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC, or des-ring B colchicine), when measured by fluorescence and enzyme activity titrations, identified the presence of a GTPase-activating protein conformational transition subsequent to the physicochemical binding of the ligands. The decrease of the apparent binding constant measured by enzyme activity in dilute buffer relative to that measured by fluorescence [for ALLO, Kb(fluor) = 1.46 x 10(6) M-1; Kb(enz act) = 1.1 x 10(5) M-1] yielded the value of the enzyme-activating conformational transition constant, K3 = 0.08.(ABSTRACT TRUNCATED AT 250 WORDS)
Perez-Ramirez
B
B
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Timasheff
S N
SN
D000818:Animals; D002417:Cattle; D003078:Colchicine / Q000031:analogs & derivatives / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D006018:Glycols / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*
D006018:Glycols; D012997:Solvents; D014404:Tubulin; D006153:Guanosine Diphosphate; D011092:Polyethylene Glycols; C032782:allocolchicine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C011480:hexylene glycol; D005990:Glycerol; D003078:Colchicine