The tryptophan synthase alpha 2 beta 2 complex. Cleavage of a flexible loop in the alpha subunit alters allosteric properties.
This study explores the catalytic and allosteric roles of a flexible loop in tryptophan synthase. Trypsin is known to cleave the tryptophan synthase alpha 2 beta 2 complex in an alpha subunit loop at Arg-188. Cleavage yields an active "nicked" alpha 2 beta 2 derivative. The new results provide evidence that the alpha subunit loop serves two important roles: substrate binding and communicating the effects of substrate binding to the beta subunit. A role for the loop in substrate binding is supported by our finding that addition of a substrate analogue of the alpha subunit, alpha-glycerol 3-phosphate, decreases the rate of cleavage by trypsin. An allosteric role for the loop is supported by the finding although the native alpha 2 beta 2 complex is strongly inhibited by alpha-glycerol 3-phosphate, the nicked alpha 2 beta 2 complex is desensitized to this inhibition. The time course of proteolysis in the presence and absence of alpha-glycerol 3-phosphate is followed by sodium dodecyl sulfate-gel electrophoresis and by assays of activity in the presence and absence of alpha-glycerol 3-phosphate. We use spectroscopic measurements of the pyridoxal phosphate-L-tryptophan intermediates at the active site of the beta subunit to determine the affinity of the native and nicked enzymes for L-tryptophan and alpha-glycerol 3-phosphate. Although cleavage alters the equilibrium distribution of intermediates and reduces the affinity for alpha-glycerol 3-phosphate, it has little effect on the affinity for amino acids bound to the beta subunit. We conclude that the loop in the alpha subunit is important for ligand binding and for communicating the effects of ligand binding from the alpha subunit to the beta subunit in the alpha 2 beta 2 complex.
Miles
E W
EW
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
D000494:Allosteric Regulation; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D012694:Serine / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012694:Serine; D014367:Tryptophan Synthase