title: Alterations of rings B and C of colchicine are cumulative in overall binding to tubulin but modify each kinetic step.
abstract: The role of the elimination of ring B and/or the modification of ring C of colchicine in tubulin binding kinetics and thermodynamics has been characterized, using four different molecules. These ligands are colchicine (COL); 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC), in which the central ring B has been reduced to one bond; allocolchicine (ALLO), in which ring C has been replaced by a six-membered ring; and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), where the same two modifications are made simultaneously. This paper describes the kinetics of association of ALLO with tubulin. The binding is accompanied by a fluorescence increase with slow biphasic kinetics, indicating binding to fast and slow tubulin isotypes. Binding to each of these isotypes occurs in two steps: a fast initial binding followed by a slower isomerization step. The K1 and k2 values for ALLO at 25 degrees C are 14,000 +/- 2,000 and 25,000 +/- 6,000 M-1 (fast and slow isotypes) and 0.055 +/- 0.003 s-1 and 0.013 +/- 0.001 s-1 (fast and slow isotype), respectively. For ALLO the reaction standard enthalpy change of the initial binding is 68 +/- 5 kJ.mol-1 (fast isotype) and 45 +/- 33 kJ.mol-1 (slow isotype) and the activation energy for the second forward step is 58 +/- 14 kJ.mol-1 (fast isotype) and 81 +/- 17 kJ.mol-1 (slow isotype). Displacement kinetics of bound ALLO by podophyllotoxin was monoexponential. The activation energy for the isomerization in the off direction is 107 +/- 7 kJ.mol-1. Comparison of the thermodynamic parameters for all four compounds shows that the modifications of both rings are cumulative with respect to overall binding. For the intermediate state there is a mutual influence of both modifications, suggesting an alteration of the reaction pathway.
authors.affiliation: Laboratory of Chemical and Biological Dynamics, Katholieke Universiteit Leuven, Belgium.
authors.forename: C
authors.initials: C
authors.lastname: Dumortier
authors.forename: M J
authors.initials: MJ
authors.lastname: Gorbunoff
authors.forename: J M
authors.initials: JM
authors.lastname: Andreu
authors.forename: Y
authors.initials: Y
authors.lastname: Engelborghs
mesh_terms: D001713:Biphenyl Compounds / Q000378:metabolism; D003078:Colchicine / Q000031:analogs & derivatives / Q000378:metabolism*; D019281:Dimerization; D005453:Fluorescence; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D011034:Podophyllotoxin / Q000494:pharmacology; D011485:Protein Binding; D013816:Thermodynamics; D014334:Tropolone / Q000031:analogs & derivatives / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
chemical_list: D001713:Biphenyl Compounds; D014404:Tubulin; C087411:2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl; C030370:2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone; C032782:allocolchicine; D014334:Tropolone; D011034:Podophyllotoxin; D003078:Colchicine