title: Subunit assembly in the tryptophan synthase alpha 2 beta 2 complex. Stabilization by pyridoxal phosphate aldimine intermediates.
abstract: This work is aimed at understanding subunit assembly in the tryptophan synthase alpha 2 beta 2 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the beta 2 subunit of tryptophan synthase for subunit association. We utilize a mutant form of the beta 2 subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T alpha 2 beta 2 complex is inactive in reactions catalyzed by the beta 2 subunit but retains activity in the reaction catalyzed by the alpha subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T beta 2 subunit and alpha 2 beta 2 complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the alpha subunit dissociates more readily from the K87T beta 2 subunit than from the wild type beta 2 subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T beta 2 subunit markedly increases the affinity for the alpha subunit and slows removal of pyridoxal phosphate by dialysis. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the C-domain in the K87T beta 2 subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type beta 2 subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type beta 2 subunit and by the external aldimine with L-serine in the K87T beta 2 subunit may further stabilize interaction with the alpha subunit because the alpha/beta interaction site contains residues from both N- and C-domains of the beta 2 subunit.
authors.affiliation: Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: U
authors.initials: U
authors.lastname: Banik
authors.forename: S A
authors.initials: SA
authors.lastname: Ahmed
authors.forename: P
authors.initials: P
authors.lastname: McPhie
authors.forename: E W
authors.initials: EW
authors.lastname: Miles
mesh_terms: D002850:Chromatography, Gel; D003956:Dialysis; D004795:Enzyme Stability; D009154:Mutation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011732:Pyridoxal Phosphate; D014367:Tryptophan Synthase