Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the a ring of bound colchicine molecule

Abstract

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of βIII in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the βIII isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different β isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all β-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on β tubulin. While the βIII isotype has two hydrophilic residues (serine²⁴² and threonine³¹⁷), both β II and β IV have two hydrophobic residues (leucine²⁴² and alanine³¹⁷). βII has isoleucine at position 318, while βIII and βIV have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.