Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Abstract

The mechanism of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK a 1 ‫؍‬ 6.5, pK a 2 ‫؍‬ 7.3, and pK a 3 ‫؍‬ 8.2-9. A significant primary isotope effect (ϳ3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the ␣-proton (␤Lys-87) is protonated at low pH, slowing the abstraction of the ␣-proton and making this step at least partially rate-limiting. pK a 2 is assigned to ␤Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK i values of 7.3. The residue with this pK a (␤Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK a values support the assignment of pK a 2 to ␤Lys-87 and suggest that the ionizing residue with pK a 1 could be a carboxylate, possibly ␤Asp-305, and that the residue associated with a conformational change at pK a 3 may be ␤Lys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.1.2.20) is a useful system for investigating relationships between protein structure and function (for reviews, see Refs. 1-4). The separate tryptophan synthase ␤ 2 subunit 1 and the ␤ subunit in the ␣ 2 ␤ 2 complex catalyze the pyridoxal phosphate (PLP) 2 -dependent conversion of L-serine and indole to L-tryptophan. A number of intermediates in this reaction have been identified from spectroscopic and kinetic studies (see Scheme I in Discussion). The three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (5) revealed that the active sites of the ␣ and ␤ subunits are ϳ25 Å apart and are connected by a hydrophobic tunnel. This tunnel serves as a passageway for indole from the active site of the ␣ subunit, where it is produced by cleavage of indole-3-glycerol phosphate, to the active site of the ␤ subunit, where it reacts with L-serine to form L-tryptophan. The activity at each site is modulated by reciprocal communication between the ␣ and ␤ sites. These heterotrophic interactions are proposed to switch the ␣ and ␤ subunits between "open" (catalytically inactive) and "closed" (catalytically active) conformations, to coordinate the activities at the two sites, and to prevent the escape of indole (4). Recent crystallographic results provide direct evidence for ligand-induced conformational changes that result in physical closure of loop structures in the ␣ subunit a...

pH Dependence of Tryptophan Synthase Catalytic Mechanism

“…It should be made clear that a single pK a does not necessarily correspond to an individual group because pK a is a group function (58). The pK a1 and pK a2 values closely correspond to the pK a1 and pK a3 values determined by investigating the pH dependence of the steady-state parameters (31). On the other hand, we do not observe the intermediate pK a2 of 7.25 that these authors detected.…”

Structure and Function of the Tryptophan Synthase α2β2 Complex

“…We have proposed previously that the open form of the ␣ 2 ␤ 2 complex, which is induced by mutation (50 -53), by solvents (54,55), or by guanidine HCl or urea (56), has increased activity in the ␤-elimination reaction relative to activity in the ␤-replacement reaction because the E-AA intermediate is more accessible to hydrolysis by solvent water in the open form and because catalysis of the ␤-replacement reaction is less efficient in the open form. Our finding of increasing ␤-elimination activity with increasing pH is consistent with the previous conclusion that the open conformation is formed at high pH (38). The H86L ␣ 2 ␤ 2 complex also showed an almost linear increase in ␤-elimination activity between pH 7.5 and 9.3, but showed much lower activity at low pH than the wildtype enzyme.…”

“…The pH profiles of the wild-type enzyme in the two reactions were very different. Although the activity was maximal at pH ϳ7.5 in the ␤-replacement reaction as reported recently (38), the activity in the ␤-elimination reaction increased between pH 6.6 and 9.2. In contrast, the activity of the H86L enzyme was very low at pH 7 and increased markedly above pH 7.5 in both reactions.…”

“…Our recent investigations of the pH dependence of the kinetic parameters of the wild-type ␣ 2 ␤ 2 complex in the ␤-replacement reaction provided evidence that Lys 87 has a pK a value of ϳ7.2, which is much lower than the pK a of the ⑀-amino group of lysine in solution (ϳ10.5) (38). Our new results and interpretation of the crystallographic data provide a plausible explanation for the low pK a of Lys 87 in the wild-type enzyme.…”

Identification of a Novel One-carbon Metabolism Regulon in Saccharomyces cerevisiae

“…High concentrations of glycine may impact upon the functioning of other enzymes involved in amino acid metabolism. For example, glycine acts as a serine analog in weak competitive inhibition of tryptophan synthase (48,49) and in non-competitive inhibition of 3-phosphoglycerate dehydrogenase, the first step of serine synthesis (50).…”