Exchange of K+ or Cs+ for Na+ induces local and long-range changes in the three-dimensional structure of the tryptophan synthase alpha2beta2 complex.

Monovalent cations activate the pyridoxal phosphate-dependent reactions of tryptophan synthase and affect intersubunit communication in the alpha2beta2 complex. We report refined crystal structures of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium in the presence of K+ at 2.0 angstrom and of Cs+ at 2.3 angstrom. Comparison of these structures with the recently refined structure in the presence of Na+ shows that each monovalent cation binds at approximately the same position about 8 angstrom from the phosphate of pyridoxal phosphate. Na+ and K+ are coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, and beta Gly-232 and to two or one water molecule, respectively. Cs+ is coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, beta Gly-232, beta Val-231, beta Gly-268 and beta Leu-304. A second binding site for Cs+ is located in the beta/beta interface on the 2-fold axis with four carbonyl oxygens in the coordination sphere. In addition to local changes in structure close to the cation binding site, a number of long-range changes are observed. The K+ and Cs+ structures differ from the Na+ structure with respect to the positions of beta Asp-305, beta Lys-167, and alpha Asp-56. One unexpected result of this investigation is the movement of the side chains of beta Phe-280 and beta Tyr-279 from a position partially blocking the tunnel in the Na+ structure to a position lining the surface of the tunnel in the K+ and Cs+ structures. The results provide a structural basis for understanding the effects of cations on activity and intersubunit communication.

Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase.

The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.

Monovalent cations affect dynamic and functional properties of the tryptophan synthase alpha 2 beta 2 complex.

Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits. In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit. Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations. The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature. We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold. The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Monovalent metal ions play an essential role in catalysis and intersubunit communication in the tryptophan synthase bienzyme complex.

This investigation shows that the alpha 2 beta 2 tryptophan synthase bienzyme complex from Salmonella typhimurium is subject to monovalent metal ion activation. The effects of the monovalent metal ions Na+ and K+ were investigated using rapid scanning stopped-flow (RSSF), single-wavelength stopped-flow (SWSF), and steady-state techniques. RSSF measurements of individual steps in the reaction of L-serine and indole to give L-trytophan (the beta-reaction) as well as the reaction of 3-indole-D-glycerol 3'-phosphate (IGP) with L-serine (the alpha beta-reaction) demonstrate that monovalent metal ions such as Na+ and K+ change the distribution of intermediates in both the transient and steady states. Therefore the metal ion effect alters relative ground-state energies and the relative positions of ground- and transition-state energies. The RSSF spectra and SWSF time courses show that the turnover of indole is significantly reduced in the absence of either Na+ or K+. The alpha-aminoacrylate Schiff base species, E(A-A), is in a less active state in the absence of monovalent metal ions. Na+ decreases the steady-state rate of IGP cleavage (the alpha-reaction) to about 30% of the value obtained in the absence of metal ions. Steady-state investigations show that in the absence of monovalent metal ions the alpha- and alpha beta-reactions have the same activity. Na+ binding gives a 30-fold stimulation of the alpha-reaction when the beta-site is in the E(A-A) form.(ABSTRACT TRUNCATED AT 250 WORDS)

Aliphatic alcohols stabilize an alternative conformation of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium.

The conformational changes that accompany association of the tryptophan synthase alpha and beta 2 subunits are probed by investigating the effects of solvents on the catalytic and spectroscopic properties of tryptophan synthase. Low concentrations of ethanol, propanol, and butanol inhibit conversion of L-serine and indole to L-tryptophan by the alpha 2 beta 2 complex. Inhibition depends on the concentration, chain length, and hydrophobicity of the alcohol. In contrast, these alcohols increase the rates of conversion of beta-chloro-L-alanine and indole to L-tryptophan and of L-serine to pyruvate. Thus, alcohols alter the substrate specificity and reaction specificity of the alpha 2 beta 2 complex. These altered specificities are similar to those of the free beta 2 subunit in aqueous solution. The spectroscopic properties of enzyme-substrate intermediates formed by the alpha 2 beta 2 complex in 3 M ethanol and 0.5 M 1-butanol also resemble the intermediates formed by the free beta 2 subunit in aqueous solution. Experiments using gel-filtration, membrane ultrafiltration, and limited proteolysis show that the alpha 2 beta 2 complex does not dissociate at low concentrations of alcohols. Our results provide evidence that alcohols can stabilize an alternative conformation of the alpha 2 beta 2 complex which is similar to the open conformation of the free beta 2 subunit in aqueous solution.