Allosteric interactions coordinate catalytic activity between successive metabolic enzymes in the tryptophan synthase bienzyme complex.
Tryptophan synthase from enteric bacteria is an alpha 2 beta 2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha 2 beta 2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Brzović
P S
PS
Department of Biochemistry, University of California, Riverside 92521-0129.
Ngo
K
K
Dunn
M F
MF
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000494:Allosteric Regulation; D002384:Catalysis; D005994:Glycerophosphates / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology; D012694:Serine / Q000378:metabolism; D013057:Spectrum Analysis; D014367:Tryptophan Synthase / Q000378:metabolism*
D005994:Glycerophosphates; D007211:Indoles; D009097:Multienzyme Complexes; C008117:indoleglycerol phosphate; D012694:Serine; C015102:dehydroalanine; D014367:Tryptophan Synthase; D000409:Alanine