BetaQ114N and betaT110V Mutations Reveal a Critically Important Role of the Substrate alpha-Carboxylte Site in the Reaction Specificity of Tryptophan Synthase
In the PLP-requiring {alpha}2{beta}2 tryptophan synthase complex, recognition of the substrate l-Ser at the {beta}-site includes a loop structure (residues {beta}110-115) extensively H-bonded to the substrate {alpha}-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: {beta}Thr110 {yields} Val, and {beta}Gln114 {yields} Asn. The {beta}T110V mutation greatly impairs both catalytic activity in the {beta}-reaction, and allosteric communication between the {alpha}- and {beta}-sites. The crystal structure of the {beta}T110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair {beta}-site catalysis and the communication of allosteric signals between the {alpha}- and {beta}-sites. Purified {beta}Q114N consists of two species of mutant protein, one with a reddish color ({lambda}max = 506 nm). The reddish species is unable to react with l-Ser. The second {beta}Q114N 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 {lambda}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 {alpha}-aminoacrylate external aldimine to the internal aldimine and {alpha}-aminoacrylate, followed by the nucleophilic attack of {alpha}-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 {beta}114-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.
- Research Organization:
- Brookhaven National Laboratory (BNL) National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- AC02-98CH10886
- OSTI ID:
- 959804
- Report Number(s):
- BNL--82790-2009-JA
- Journal Information:
- Biochemistry, Journal Name: Biochemistry Vol. 46; ISSN 0006-2960; ISSN BICHAW
- Country of Publication:
- United States
- Language:
- English
Similar Records
Structural Snapshots of Proteus vulgaris Tryptophan Indole-Lyase Reveal Insights into the Catalytic Mechanism
Structure of soybean [beta]-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants