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. Naf binding gives a 30-fold stimulation of the alpha-reaction when the beta-site is in the E(A-A) form. This study demonstrates that the presence of saturating amounts of monovalent metal ions is an important element both for catalysis at the beta-site and for the allosteric linkage between the alpha- and beta-sites in the bienzyme complex. Theoretical models that describe the monovalent metal ion effect are discussed and tested with the help of computer simulation.