Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule.
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 beta(III) 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 beta(III) 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 beta 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 beta-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 beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) 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.
Gupta
Suvroma
S
Department of Biochemistry, Bose Institute, Centenary Campus, Calcutta 700 054, India.
Banerjee
Mithu
M
Poddar
Asim
A
Banerjee
Asok
A
Basu
Gautam
G
Roy
Debjani
D
Bhattacharyya
Bhabatarak
B
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002417:Cattle; D003078:Colchicine / Q000737:chemistry* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007532:Isoleucine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D014633:Valine / Q000737:chemistry
D000596:Amino Acids; D010446:Peptide Fragments; D020033:Protein Isoforms; D014404:Tubulin; D007532:Isoleucine; D007930:Leucine; D014633:Valine; D000409:Alanine; D003078:Colchicine