Nueleophile specificity in anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and salicylate synthase

Kristin T. Ziebart, Michael D. Toney

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Anthranilate synthase (AS), aminodeoxychorismate synthase (ADCS), isochorismate synthase (IS), and salicylate synthase (SS) are structurally homologous chorismate-utilizing enzymes that carry out the first committed step in the formation of tryptophan, folate, and the siderophores enterobactin and mycobactin, respectively. Each enzyme catalyzes a nucleophilic substitution reaction, but IS and SS are uniquely able to employ water as a nucleophile. Lys147 has been proposed to be the catalytic base that activates water for nucleophilic attack in IS and SS reactions; in AS and ADCS, glutamine occupies the analogous position. To probe the role of Lys147 as a catalytic base, the K147Q IS, K147QSS, Q147K AS, and Q147K ADCS mutants were prepared and enzyme reactions were analyzed by high-performance liquid chromatography. Q147K AS employs water as a nucleophile to a small extent, and the cognate activities of K147Q IS and K147Q SS were reduced ∼25- and ∼50-fold, respectively. Therefore, Lys147 is not solely responsible for activation of water as a nucleophile. Additional factors that contribute to water activation are proposed. A change in substrate preference for K147Q SS pyruvate lyase activity indicates Lys147 partially controls SS reaction specificity. Finally, we demonstrate that AS, ADCS, IS, and SS do not possess chorismate mutase promiscuous activity, contrary to several previous reports.

Original languageEnglish (US)
Pages (from-to)2851-2859
Number of pages9
JournalBiochemistry
Volume49
Issue number13
DOIs
StatePublished - Apr 6 2010

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Anthranilate Synthase
Salicylates
Nucleophiles
Water
Chorismate Mutase
Pyruvate Synthase
Enzymes
Enterobactin
Chemical activation
Siderophores
Lyases
High performance liquid chromatography
isochorismate synthase
aminodeoxychorismate synthase
Glutamine
Folic Acid
Tryptophan
Substitution reactions
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Nueleophile specificity in anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and salicylate synthase. / Ziebart, Kristin T.; Toney, Michael D.

In: Biochemistry, Vol. 49, No. 13, 06.04.2010, p. 2851-2859.

Research output: Contribution to journalArticle

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abstract = "Anthranilate synthase (AS), aminodeoxychorismate synthase (ADCS), isochorismate synthase (IS), and salicylate synthase (SS) are structurally homologous chorismate-utilizing enzymes that carry out the first committed step in the formation of tryptophan, folate, and the siderophores enterobactin and mycobactin, respectively. Each enzyme catalyzes a nucleophilic substitution reaction, but IS and SS are uniquely able to employ water as a nucleophile. Lys147 has been proposed to be the catalytic base that activates water for nucleophilic attack in IS and SS reactions; in AS and ADCS, glutamine occupies the analogous position. To probe the role of Lys147 as a catalytic base, the K147Q IS, K147QSS, Q147K AS, and Q147K ADCS mutants were prepared and enzyme reactions were analyzed by high-performance liquid chromatography. Q147K AS employs water as a nucleophile to a small extent, and the cognate activities of K147Q IS and K147Q SS were reduced ∼25- and ∼50-fold, respectively. Therefore, Lys147 is not solely responsible for activation of water as a nucleophile. Additional factors that contribute to water activation are proposed. A change in substrate preference for K147Q SS pyruvate lyase activity indicates Lys147 partially controls SS reaction specificity. Finally, we demonstrate that AS, ADCS, IS, and SS do not possess chorismate mutase promiscuous activity, contrary to several previous reports.",
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