SUBUNIT ASSEMBLY IN THE TRYPTOPHAN SYNTHASE ALPHA(2)BETA(2) COMPLEX - STABILIZATION BY PYRIDOXAL-PHOSPHATE ALDIMINE INTERMEDIATES

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. me 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 Pa subunit and by the external aldimine with L-serine in the K87T beta(2) subunit may further stabilize interaction with the iv subunit because the alpha/beta interaction site contains residues from both N- and C-domains of the beta(2) subunit.