title: Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.
abstract: Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzović, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed" structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.
authors.affiliation: Department of Biochemistry, University of California, Riverside 92521-0129.
authors.forename: P S
authors.initials: PS
authors.lastname: Brzović
authors.forename: Y
authors.initials: Y
authors.lastname: Sawa
authors.forename: C C
authors.initials: CC
authors.lastname: Hyde
authors.forename: E W
authors.initials: EW
authors.lastname: Miles
authors.forename: M F
authors.initials: MF
authors.lastname: Dunn
mesh_terms: D000494:Allosteric Regulation; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D007211:Indoles; C057812:indoline; C030374:indole; D004247:DNA; D014367:Tryptophan Synthase