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

Abstract

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.

Biochemical and Structural Insights into Microtubule Perturbation by CopN from Chlamydia pneumoniae

“…We studied the interference of pCopN with tubulin nucleotide exchange by recording the quenching of the fluorescence of tubulin's tryptophan residues upon substitution of GDP by S6-GDP (20,21). Fitting the data with a monoexponential decay function, we found a rate constant of 0.15 Ϯ 0.02 s Ϫ1 for tubulin alone, in agreement with previous results (21).…”

Stathmin Slows down Guanosine Diphosphate Dissociation from Tubulin in a Phosphorylation-Controlled Fashion

“…The rate of GTP dissociation from the 1:1 GTP-tubulin complex by excess S6-GTP was found to be 10-fold slower (0.012 s -1 ) under the same solution conditions. The dependence of the rate of dissociation of GDP on solution variables showed that glycerol (4 M) caused a 10fold decrease in the rate of GDP dissociation, in qualitative agreement with Yarbrough and Fishback (27). The rate of GDP dissociation increased (k ) 0.3 s -1 ) when the concentration of magnesium ions in P buffer was increased to 5 mM.…”

“…Kinetics of GDP and GTP Dissociation from Tubulin in the Absence and Presence of Stathmin. The fluorescence decrease associated with the fluorescence resonance energy transfer from tryptophans to S6-GDP ( , ) was used to monitor the kinetics of S6-GDP exchange for tubulin-bound GDP and to examine how the kinetics were affected by the binding of stathmin to tubulin. GDP-tubulin 1:1 complex was first prepared by resuspending the pellet of precycled microtubules in ice-cold M buffer supplemented with 20 μM GDP.…”

GDP to GTP exchange on the microtubule end can contribute to the frequency of catastrophe

“…4B). A prior measurement of GTP binding to yeast αβ-tubulin showed higher affinity (~50 nM; (Davis et al, 1993)); this discrepancy may be explained by the glycerol-free conditions we used (glycerol has been observed to increase the nucleotide binding affinity of αβ-tubulin (Yarbrough and Fishback, 1985)). Whatever the reason, our measurements showed that the β:C12A mutation caused a decrease in the affinity of GTP/GDP binding.…”