Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex.
Allosteric interactions regulate substrate channeling in Salmonella typhimurium tryptophan synthase. The channeling of indole between the alpha- and beta-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the alpha-site, and covalent reaction of l-Ser at the beta-site. This signaling switches the alpha- and beta-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of alpha-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3'-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of alpha-site-beta-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine complexed with G3P and with three of the new ASLs are presented. These structural and solution studies of the ASL complexes with the internal aldimine form of the enzyme establish the following. (1) ASL binding occurs with high specificity and relatively high affinities at the alpha-site. (2) Binding of the new ASLs slows the entry of indole analogues into the beta-site by blocking the tunnel opening at the alpha-site. (3) ASL binding stabilizes the closed conformations of the beta-subunit for the alpha-aminoacrylate and quinonoid forms of the enzyme. (4) The new ASLs exhibit allosteric properties that parallel the behaviors of IGP and G3P.
Ngo
Huu
H
Department of Biochemistry, University of California, Riverside, California 92521, USA.
Harris
Rodney
R
Kimmich
Novelle
N
Casino
Patricia
P
Niks
Dimitri
D
Blumenstein
Lars
L
Barends
Thomas Reinier
TR
Kulik
Victor
V
Weyand
Michael
M
Schlichting
Ilme
I
Dunn
Michael F
MF
D000494:Allosteric Regulation / Q000502:physiology*; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001665:Binding Sites; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology; D012694:Serine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D005994:Glycerophosphates; D005986:Glyceraldehyde 3-Phosphate; C008117:indoleglycerol phosphate; D012694:Serine; C027132:1-anilino-8-naphthalenesulfonate; D014367:Tryptophan Synthase