The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

Abstract

The a subunit is bound with negative cooperativity to the holo(2 subunit of tryptophan synthase in phosphate buffer.Thus it is feasible to measure separately the rates of formation both of the stable aC32 subcomplex from O2, and of the mature a2f32 complex from rn2, using stopped-flow techniques. Addition of each a subunit proceeds in two steps; an initial cx, protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the 0a2 and a2/2 complexes were measured by trapping released a subunit with enzymically inactive reduced (2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the (3 protomer for the a subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second a subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-erine and indolepropanol phosphate during the assembly process shows that the (3 protomer is already partially activated in the initial a(3 complex. Full activation is achieved in the slow isomerization reaction. In contrast, the a subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction.These observations indicate that the isomerization involves synchronous conformation changes of both a and (3 protomers.

On the Role of Helix 0 of the Tryptophan Synthetase α Chain of

“…Considerable evidence exists demonstrating that in the TSase enzyme complex the active sites of the enzyme subunits are cross-activated by bound substrates (35,36) and that substrate binding increases subunit association (34 -36). L-Serine, the ligand of TrpB, following conversion to an aminoacrylate intermediate, is believed to induce a conformational change in TrpB that is communicated to the active site of TrpA, leading to activation of cleavage of InGP at the TrpA active site (35,36).…”

“…L-Serine, the ligand of TrpB, following conversion to an aminoacrylate intermediate, is believed to induce a conformational change in TrpB that is communicated to the active site of TrpA, leading to activation of cleavage of InGP at the TrpA active site (35,36). Similarly, InGP, the substrate of TrpA, increases TrpA's association with TrpB and greatly stimulates TrpB's activity in the indole ϩ serine 3 tryptophan reaction (36). ␣-Glycerophosphate, a TrpA substrate analog, also can increase TrpB enzymatic activity.…”

“…The addition of indolepropanol phosphate or ␣-glycerophosphate, ligands that bind to TrpA, increased both the affinity of the mutant TrpAs for TrpB and TrpB enzymatic activity. These ligands were proposed to increase TrpB activity by inducing conformational changes in TrpA that affect interaction with TrpB (36). It has also been shown that ligands such as InGP that bind to TrpA can reverse the deleterious effects of an amino acid substitution in TrpB that alters the contact region with TrpA (38).…”

A Thermodynamic Analysis of Conformational Change Due to the α2β2Complex Formation of Tryptophan Synthase

“…The mutual activation is considered to result from conformational changes in both subunits induced by subunit association. The conformational change in both subunits upon complex formation has been suggested by kinetic studies (Lane et al, 1984), calorimetry (Wiesinger et al, 1979), small-angle X-ray scattering (Wilhelm et al, 1982), fluorescence and hydrodynamic measurements (Lane, 1983 ;Lane et al, 1984;Lane and Kirschner, 1983), neutron scattering (Ibel et al, 1985), and immunochemical studies with monoclonal antibodies against the j? subunit (Murry-Brelier and Goldberg, 1990).…”

Kinetic Characterization of Channel Impaired Mutants of Tryptophan Synthase

“…It has previously been shown that there are absorbance and fluorescence changes exhibited by the pyridoxal phosphate upon reaction with serine and chemical nature of the serine-pyridoxal phosphate complexes have been defined (Faeder and Hammes, 1971;York, 1972;Lane and Kirschner, 1983;Lane et al, 1984;Drewe and Dunn, 1985). In the first step, serine displaces the internal aldimine formed between Lys-87 and the pyridoxal phosphate at the active site of the ␤ subunit to form the external aldimine (E-Ser in Schemes II and III), which is highly fluorescent, with emission at 500 nm upon excitation at 405 nm.…”