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 k(cat) with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease K-M for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the k(cat)(-)/K-M 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. In the absence of cations, at pH 7.9, the predominant species is the alpha-aminoacrylate absorbing at 350 nm; Cs+, Rb+, and Li+ cause the formation of a new absorption at about 470 nm, tentatively assigned to a tautomer of the alpha-aminoacrylate, whereas Na+ and K+ stabilize the external aldimine absorbing at 422 nm. By single-crystal polarized absorption microspectrophotometry, we verified that the cation-specific effects on the equilibrium distribution of beta-subunit intermediates are maintained in the crystalline state. These studies help to define the experimental conditions suitable for X-ray crystallographic studies aiming to identify the cation-binding sites and their mode of action.