High-pressure dissociation of the beta 2-dimer of tryptophan synthase from Escherichia coli monitored by sucrose gradient centrifugation.
The isolated beta 2-dimer of Escherichia coli tryptophan synthase exhibits reversible high-pressure deactivation and hybridization with an equilibrium transition at 690 and 870 bar for the apoenzyme and holoenzyme, respectively. To investigate the hypothetical dissociation mechanism ultracentrifugal analysis has been applied. In a conventional swing-out rotor (r(max) = 16 cm, fill-height 9 cm) a pressure gradient of 1 less than p less than 1840 bar is formed at maximum speed (40 000 rpm). Using a sucrose gradient to stabilize the particle distribution, pressure-dependent alterations of the state of association of oligomeric systems may be determined. In the present experiments ovalbumin (with a molecular mass close to the beta-monomer) has been used as a reference. The radial sedimentation velocity of the beta 2-dimer (in 5-20% sucrose, 10 degrees C) is found to decrease significantly at p approximately equal to 850 bar. From the slopes in an r-r(degrees) vs t plot the limiting values for the particle weight at the meniscus and the bottom of the tube are found to be the beta 2-dimer (M(r) = 85 800) and the beta-monomer (M(r) = 42 900), thus proving pressure-dependent dissociation. Since sucrose stabilizes the native quaternary structure, the beta 2 leads to 2 beta transition is shifted towards higher pressures compared to the dissociation in standard buffer. Conventional quench experiments in high-pressure cells in the presence of 13% (w/v) sucrose confirm the result of the sucrose gradient centrifugation with respect to the critical pressure where deactivation (and dissociation) occur.
D002499:Centrifugation, Density Gradient / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D011312:Pressure; D014367:Tryptophan Synthase / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D014367:Tryptophan Synthase