Subunit Assembly in the Tryptophan Synthase α2β2 Complex

• Published in: The Journal of biological chemistry 1995-04, Vol.270 (14), p.7944-7949
• Main Authors: Banik, Utpal, Ahmed, S. Ashraf, McPhie, Peter, Miles, Edith Wilson
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.14.7944

This work is aimed at understanding subunit assembly in the tryptophan synthase α2β2 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the β2 subunit of tryptophan synthase for subunit association. We utilize a mutant form of the β2 subunit that is unable to form the internal aldimine because lysine 87 is replaced by threonine (K87T). The K87T α2β2 complex is inactive in reactions catalyzed by the β2 subunit but retains activity in the reaction catalyzed by the α subunit. We find that dialysis removes pyridoxal phosphate much more rapidly from the K87T β2 subunit and α2β2 complex than from the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the α subunit dissociates more readily from the K87T β2 subunit than from the wild type β2 subunit. The reaction of L-serine to form an external aldimine with pyridoxal phosphate at the active site of the K87T β2 subunit markedly increases the affinity for the α 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 β2 subunit. This interdomain bridge may mimic the internal aldimine bond in the wild type β2 subunit and stabilize pyridoxal phosphate binding. The interdomain bridges formed by the internal aldimine with the wild type β2 subunit and by the external aldimine with L-serine in the K87T β2 subunit may further stabilize interaction with the α subunit because the α/β interaction site contains residues from both N- and C-domains of the β2 subunit.