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) subu nit of tryptophan synthase for subunit association. We utilize a mutan t form of the beta(2) subunit that is unable to form the internal aldi mine 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 su bunit. We find that dialysis removes pyridoxal phosphate much more rap idly from the K87T beta(2) subunit and alpha(2) beta(2) complex than f rom the wild type counterparts. Activity measurements, gel filtration, and subunit interchange experiments show that the alpha subunit disso ciates more readily from the K87T beta(2) subunit than from the wild t ype beta(2) subunit. The reaction of L-serine to form an external aldi mine with pyridoxal phosphate at the active site of the K87T beta(2) s ubunit markedly increases the affinity for the alpha subunit and slows removal of pyridoxal phosphate by dialysis. me propose that the exter nal aldimine between L-serine and pyridoxal phosphate bridges the N-do main and the C-domain in the K87T beta(2) subunit. This interdomain br idge may mimic the internal aldimine bond in the wild type beta(2) sub unit and stabilize pyridoxal phosphate binding. The interdomain bridge s 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 f urther stabilize interaction with the iv subunit because the alpha/bet a interaction site contains residues from both N- and C-domains of the beta(2) subunit.