D. Ferrari et al., beta D305A mutant of tryptophan synthase shows strongly perturbed allosteric regulation and substrate specificity, BIOCHEM, 40(25), 2001, pp. 7421-7432
Substrate channeling in the tryptophan synthase bienzyme is regulated by al
losteric interactions. Allosteric signals are transmitted via a scaffolding
of structural elements that includes a monovalent cation-binding site and
salt-bridging interactions between the side chains of beta Asp 305, beta Ar
g 141, beta Lys 167, and alpha Asp 56 that appear to modulate the interconv
ersion between open and closed conformations. beta Asp 305 also interacts w
ith the hydroxyl group of the substrate L-Ser in some structures. One possi
ble functional role for beta Asp 305 is to ensure the allosteric transmissi
on that triggers the switching of alpha beta -dimeric units between open an
d closed conformations of low and high activity. This work shows that subst
itution of beta Asp 305 with Ala (beta D305A) decreases the affinity of the
beta -site for the substrate L-Ser, destabilizes the enzyme-bound alpha -a
minoacrylate, E(A-A), and quinonoid species, E(Q), and changes the nucleoph
ile specificity of the beta -reaction. The altered specificity provides a b
iosynthetic route for new L-amino acids reaction with L-Ser relative to the
wild-type enzyme. The formation of pyruvate is strongly inhibited by the b
inding of benzimidazole to E(A-A). Upon reaction with L-Ser and in the pres
ence of the alpha -site substrate analogue, alpha -glycerol phosphate, the
Na+ form of beta D305A undergoes inactivation via reaction of nascent alpha
-aminoacrylate with bound PLP. This work establishes important roles for b
eta Asp 305 both in the conformational change between open and closed state
s that takes place at the beta -site during the formation of the E(A-A) and
in substrate binding and recognition.