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Abstract

In the PLP-requiring α2α2 tryptophan synthase complex, recognition of the substrate L-Ser at the β-site includes a loop structure (residues β110-115) extensively H-bonded to the substrate α-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: βThr110 Val, and βGln114 Asn. The βT110V mutation greatly impairs both catalytic activity in the β-reaction, and allosteric communication between the α- and β-sites. The crystal structure of the βT110V mutant shows that the modified L-Ser carboxylate subsite has altered protein interactions that impair β-site catalysis and the communication of allosteric signals between the α- and β-sites. Purified βQ114N consists of two species of mutant protein, one with a reddish color (max = 506 nm). The reddish species is unable to react with L-Ser. The second βQ114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate L-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with L-Ser results in the formation of an inactive species during the first 15 min with max ˜ 320 nm, followed by a slower conversion over 24 h to the species with max = 506 nm. The 320 and 506 nm species originate from conversion of the α-aminoacrylate external aldimine to the internal aldimine and α-aminoacrylate, followed by the nucleophilic attack of α-aminoacrylate on C-4'; of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4'; to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of β114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase.