Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli

Abstract

For the a subunit of tryptophan synthase and at constant concentration of D-glyceraldehyde 3-phosphate the saturation curves with respect to indole concentration are weakly sigmoidal. This phenomenon can be explained by interaction between indole bound to the effector site established previously and the active center of the monomeric a subunit. Kinetic studies of the inhibition of indoleglycerol phosphate synthesis by the analogue indolepropanol phosphate show that the inhibition is competitive with respect to D-glyceraldehyde 3-phosphate and non-competitive with respect to indole. Mechanisms with random addition of substrates or ordered addition with indole binding first can therefore be excluded. A quantitative fit of the data has been obtained to an ordered addition mechanism with D-glyceraldehyde 3-phosphate binding first and with a distribution of the enzyme between two states differing in V, governed by the binding of indole to the effector site.The kinetic constants obtained for the a subunit have been compared with those of the a& complex of tryptophan synthase. Protein-protein interaction of the a subunit with the subunit (a) does not alter the catalytic mechanism of indoleglycerol phosphate synthesis, (b) suppresses the substrate activation by indole, and (c) changes the various equilibrium, rate and steady-state constants in the sense of conveying higher substrate specificity and catalytic efficiency to the a-subunit. The ocurrence of local and gross conformational changes in the tryptophan synthase system is discussed.Work in this laboratory is concerned with understanding the effect of protein-protein interaction on the intrinsiic properties of the component subunits of tryptophan synthase of Escherichia coli. In the preceding paper we have delineated the mechanism by which the native complex of tryptophan synthase catalyzes 1 he synthesis of indoleglycerol phosphate P I .This reaction is one of the three reactions catalyzed by the multi-enzyme complex. It proceeds 100 times more slowly when an equivalent amount of the isolated a subunit is the catalyst [2]. It is pertinent to enquire whether this quantitative difference is due to a change in the basic catalytic mechanism. Little is known about the steady-state kinetics of the a subunit [3]. The mechanism by which the a subunit catalyzes the synthesis + Indoleglycerol phosphate

The Role of Oligomerization and Cooperative Regulation in Protein Function: The Case of Tryptophan Synthase

“…The value suggests that the transition rate may be decreased by several orders of magnitude in the isolated α–monomer but reduced only a little in the isolated β–monomer. The results are in good agreement with experiments showing that the catalytic rate is ∼100 times slower in the isolated α–monomer but only 1.5 times slower in the isolated β–monomer as compared with the αββα tetramer [28]–[29]. The calculation further supports our conjecture that one major role of oligomerization in TRPS is to help the ligand binding processes.…”

“…Steady–state kinetic studies [28] revealed that the rate of the α–reaction in the isolated α–subunit is ∼100 times slower than that in the αββα tetramer of Escherichia coli TRPS, which has 84% identities and 94% similarities with the Salmonella typhimurium TRPS used in our simulation studies. This observation reflects a strong synergistic effect of subunits on the α–catalysis in the multi-enzyme complex.…”

Directed evolution of ( )8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase -subunit

“…TrpA_1-2 has the highest k cat /K M PRA value of all analysed variants (Table III). We tested whether this variant is still able to catalyse the reverse TrpA reaction (synthesis of IGP from indole and D-glyceraldehyde 3-phosphate) (Weischet and Kirschner, 1976), which is thermodynamically preferred to the physiological reaction (Fig. 1).…”

Severing of a hydrogen bond disrupts amino acid networks in the catalytically active state of the alpha subunit of tryptophan synthase

“…indole and G3P react to form IGP). It should be noted that the catalytic activity of the free αTS enzyme is lower than when in found in complex with the βTS subunit . The T183V substitution also led to a substantial decrease in k cat , but had little effect on the K M for indole or G3P (Table ).…”