The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.
The alpha subunit is bound with negative cooperativity to the holo beta 2 subunit of tryptophan synthase in phosphate buffer. Thus it is feasible to measure separately the rates of formation both of the stable alpha beta 2 subcomplex from beta 2, and of the mature alpha 2 beta 2 complex from alpha beta 2, using stopped-flow techniques. Addition of each alpha subunit proceeds in two steps; an initial alpha beta protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the alpha beta 2 and alpha 2 beta 2 complexes were measured by trapping released alpha subunit with enzymically inactive reduced beta 2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the beta protomer for the alpha 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 alpha subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-serine and indolepropanol phosphate during the assembly process shows that the beta protomer is already partially activated in the initial alpha beta complex. Full activation is achieved in the slow isomerization reaction. In contrast, the alpha subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction. These observations indicate that the isomerization involves synchronous conformation changes of both alpha and beta protomers.
D001426:Bacterial Proteins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D011487:Protein Conformation; D012694:Serine / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000096:biosynthesis*
D001426:Bacterial Proteins; D007211:Indoles; C041272:indolepropanol phosphate; D012694:Serine; D014367:Tryptophan Synthase