Reconstitution of the isolated beta2-subunit of tryptophan synthase from Escherichia coli after dissociation induced by high hydrostatic pressure. Equilibrium and kinetic studies.

The isolated beta2-subunit of Escherichia coli tryptophan synthase can be reversibly dissociated into enzymically inactive monomers under high hydrostatic pressure. Deactivation at 1.5 kbar which shows a half-time of 11 min (rate constant k=10 (-3) s (-1) is paralleled by dissociation with a small lag phase of about 5 min. Pressure release leads to 95 +/- 5% recovery of specific activity and complete restoration of the hydrodynamic and spectral properties which specify the native dimer. Over the concentration range 1-100 micrograms/ml (0.02-2.3 micrograms M) the kinetics of reactivation can be fitted by one apparent first-order rate constant (k=6.5 +/- 0.6 X 10 (-4) s (-1), half-time = 17.5 min). The reconstitution of catalytic activity is paralleled by alterations in tryptophan fluorescence at 327 nm, thus presenting direct evidence for conformational changes in the direct vicinity of the active center (k1 = 1.9 X 10 (-3) s(-1), k2 = 6.5 +/- 0.6 X 10(-4) s (-1) ). On the other hand, a definite mechanism of reactivation requires the association of the refolding monomers to be included. The kinetics of dimerization have been followed via hybridization between native and chemically modified beta-chains, yielding an apparent first-order rate constant of 6.3 +/- 0.6 X 10 (-4) s (-1). As a consequence, we propose a sequential uni-uni-bimolecular mechanism, which is characterized by a minimum of two conformational changes in substantially structured monomers followed by a fast dimerization reaction to yield the active beta2-subunit.