title: Microspectrophotometric studies on single crystals of the tryptophan synthase alpha 2 beta 2 complex demonstrate formation of enzyme-substrate intermediates.
abstract: Microspectrophotometry of single crystals of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium is used to compare the catalytic and regulatory properties of the enzyme in the soluble and crystalline states. Polarized absorption spectra demonstrate that chromophoric intermediates are formed between pyridoxal phosphate at the active site of the beta subunit and added substrates, substrate analogs, and reaction intermediate analogs. Although the crystalline and soluble forms of the enzyme produce some of the same enzyme-substrate intermediates, including Schiff base and quinonoid intermediates, in some cases the equilibrium distribution of these intermediates differs in the two states of the enzyme. Ligands which bind to the active site of the alpha subunit alter the distribution of intermediates formed at the active site of the beta subunit in both the crystalline and soluble states. The three-dimensional structures of the tryptophan synthase alpha 2 beta 2 complex and of a derivative with indole-3-propanol phosphate bound at the active site of the alpha subunit have recently been reported (Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) J. Biol. Chem. 264, 17857-17871). Our present findings help to establish experimental conditions for selecting defined intermediates for future x-ray crystallographic analysis of the alpha 2 beta 2 complex with ligands bound at the active sites of both alpha and beta subunits. These crystallographic studies should explain how catalysis occurs at the active site of the beta subunit and how the binding of a ligand to one active site affects the binding of a ligand to the other active site which is 25 A away.
authors.affiliation: Institute of Biochemical Sciences, University of Parma, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Mozzarelli
authors.forename: A
authors.initials: A
authors.lastname: Peracchi
authors.forename: G L
authors.initials: GL
authors.lastname: Rossi
authors.forename: S A
authors.initials: SA
authors.lastname: Ahmed
authors.forename: E W
authors.initials: EW
authors.lastname: Miles
mesh_terms: D003460:Crystallization; D046911:Macromolecular Substances; D011048:Polarography; D011485:Protein Binding; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000494:pharmacology; D013053:Spectrophotometry; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D012694:Serine; D014364:Tryptophan; D014367:Tryptophan Synthase