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.
Highlights
This work is aimed at understanding subunit assembly in the tryptophan synthase 0:2132 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the 132 subunit of tryptophan synthase for subunit association
The interdomain bridges formed by the internal aldimine with the wild type 132 subunit and by the external aldimine with t-serine in the K87T 132 subunit may further stabilize interaction with the 0:subunit because the cxI13 interaction site contains residues from both N- and Codomains of the 132 subunit
Formation of the internal aldimine with lysine 87 in the wild type enzymes stabilizes the enzymes to loss of pyridoxal phosphate upon dialysis
Summary
This work is aimed at understanding subunit assembly in the tryptophan synthase 0:2132 complex and the importance of the internal aldimine between pyridoxal phosphate and lysine 87 of the 132 subunit of tryptophan synthase for subunit association. We propose that the external aldimine between L-serine and pyridoxal phosphate bridges the N-domain and the Codomain in the K87T 132 subunit This interdomain bridge may mimic the internal aldimine bond in the wild type 132 subunit and stabilize pyridoxal phosphate binding. In the present work we ask whether binding pyridoxal phosphate per se increases subunit affinity or whether formation of the internal aldimine with 132 subunit lysine 87 is required. To answer this question, we use a mutant form of the {32 subunit having the lysine 87 replaced by threonine (K87T) [10]. The results presented here provide evidence that formation of interdomain bridges by pyridoxal phosphate-aldimine intermediates stabilizes subunit association and stabilizes pyridoxal phosphate binding
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