Kinetics of interaction of 2-amino-6-mercapto-9-.beta.-ribofuranosylpurine 5'-triphosphate with bovine brain tubulin

• Published in: Biochemistry (Easton) Vol. 24; no. 7; pp. 1708 - 1714
• Main Authors: Yarbrough, Lynwood R, Fishback, James L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-03-1985
• Subjects: Animals; Binding Sites; Binding, Competitive; Biological and medical sciences; Brain - metabolism; Cattle; Fundamental and applied biological sciences. Psychology; Glycerol - pharmacology; Guanosine Diphosphate - metabolism; Guanosine Triphosphate - analogs & derivatives; Guanosine Triphosphate - metabolism; In Vitro Techniques; Interactions. Associations; Intermolecular phenomena; Kinetics; Molecular biophysics; Protein Conformation; Spectrometry, Fluorescence; Thionucleotides - metabolism; Tubulin - metabolism; Molecular association; Contractile protein; Tubulin; Kinetics; Fluorescence spectrometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00328a021

The binding of the guanine nucleotide analogue 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-triphosphate (S6-GTP) to tubulin from which the associated proteins and exchangeably bound nucleotide have been removed produces about a 15% decrease in intrinsic tubulin fluorescence. Using a fluorescence stopped-flow technique, we have examined the kinetics and mechanism of this process. Analysis of the data reveals that the binding is complex, involving at least one conformational change subsequent to nucleotide binding. The bimolecular association rate constant for binding of S6-GTP to tubulin is approximately 6 X 10(5) M-1 s-1, suggesting that the orientation requirements are stringent. The kinetic parameters for dissociation of GDP, S6-GTP, and S6-GDP from the exchangeable nucleotide binding site have also been determined. S6-GDP and GDP were found to have comparable rates of dissociation; S6-GTP dissociated approximately twice as slowly as either GDP or S6-GDP. Glycerol produces a significant decrease in the rates of nucleotide dissociation. The mechanism whereby glycerol produces such an effect is not known; however, it may involve slight changes in the conformation of the tubulin protomer.