The cofactors role on chemical mechanism of recombinant acetohydroxy acid synthase from tobacco

Joungmok Kim, Jung Rim Kim, Young Tae Kim, Jung Do Choi, Moon Young Yoon

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. The active site contains several conserved ionizable groups and all of these appear to be important as judged by the fact that mutation diminishes or abolishes catalytic activity. Recently, we have shown [Yoon, M.-Y., Hwang, J.-H., Choi, M.-K., Baek, D.-K., Kim, J., Kim, Y.-T., Choi, J.-D. FEBS Letters 555 (2003), 185-191] that the activity is pH-dependent due to changes in Vmax and V/K m. Data were consistent with a mechanism in which substrate was selectively catalyzed by the enzyme with an unprotonated base having a pK 6.48, and a protonated group having a pK of 8.25 for catalysis. Here, we have in detail studied the pH dependence of the kinetic parameters of the cofactors (ThDP, FAD, Mg2+) in order to obtain information about the chemical mechanism in the active site. The Vmax of kinetic parameters for all cofactors was pH-dependent on the basic side. The pK of ThDP, FAD and Mg2+ was 9.5, 9.3 and 10.1, respectively. The V/Km of kinetic parameters for all cofactors was pH-dependent on the acidic and on the basic side. The pK of ThDP, FAD and Mg2+ was 6.2-6.4 on the acidic side and 9.0-9.1 on the basic side. The well-conserved histidine mutant (H392) did not affect the pH-dependence of the kinetic parameters. The data are discussed in terms of the acid-base chemical mechanism.

Original languageEnglish
Pages (from-to)721-725
Number of pages5
JournalBulletin of the Korean Chemical Society
Volume25
Issue number5
DOIs
StatePublished - 2004 May 20

Fingerprint

Acetolactate Synthase
Tobacco
Kinetic parameters
Flavin-Adenine Dinucleotide
Thiamine Pyrophosphate
Divalent Cations
Enzymes
Histidine
Catalysis
Catalyst activity
Acids
Substrates

Keywords

  • Acetohydroxy acid synthase
  • Chemical mechanism
  • Cofactors
  • PH study

Cite this

Kim, Joungmok ; Kim, Jung Rim ; Kim, Young Tae ; Choi, Jung Do ; Yoon, Moon Young. / The cofactors role on chemical mechanism of recombinant acetohydroxy acid synthase from tobacco. In: Bulletin of the Korean Chemical Society. 2004 ; Vol. 25, No. 5. pp. 721-725.
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abstract = "Acetohydroxy acid synthase (AHAS) is one of several enzymes that require thiamine diphosphate and a divalent cation as essential cofactors. The active site contains several conserved ionizable groups and all of these appear to be important as judged by the fact that mutation diminishes or abolishes catalytic activity. Recently, we have shown [Yoon, M.-Y., Hwang, J.-H., Choi, M.-K., Baek, D.-K., Kim, J., Kim, Y.-T., Choi, J.-D. FEBS Letters 555 (2003), 185-191] that the activity is pH-dependent due to changes in Vmax and V/K m. Data were consistent with a mechanism in which substrate was selectively catalyzed by the enzyme with an unprotonated base having a pK 6.48, and a protonated group having a pK of 8.25 for catalysis. Here, we have in detail studied the pH dependence of the kinetic parameters of the cofactors (ThDP, FAD, Mg2+) in order to obtain information about the chemical mechanism in the active site. The Vmax of kinetic parameters for all cofactors was pH-dependent on the basic side. The pK of ThDP, FAD and Mg2+ was 9.5, 9.3 and 10.1, respectively. The V/Km of kinetic parameters for all cofactors was pH-dependent on the acidic and on the basic side. The pK of ThDP, FAD and Mg2+ was 6.2-6.4 on the acidic side and 9.0-9.1 on the basic side. The well-conserved histidine mutant (H392) did not affect the pH-dependence of the kinetic parameters. The data are discussed in terms of the acid-base chemical mechanism.",
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The cofactors role on chemical mechanism of recombinant acetohydroxy acid synthase from tobacco. / Kim, Joungmok; Kim, Jung Rim; Kim, Young Tae; Choi, Jung Do; Yoon, Moon Young.

In: Bulletin of the Korean Chemical Society, Vol. 25, No. 5, 20.05.2004, p. 721-725.

Research output: Contribution to journalArticle

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