Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

Virinchipuram Viswanathan, J. M. Green, B. P. Nichols

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB undergoes inactivation upon incubation at 37°C, which is prevented by the presence of chorismate or PabA; glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the K(m) values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.

Original languageEnglish (US)
Pages (from-to)5918-5923
Number of pages6
JournalJournal of Bacteriology
Volume177
Issue number20
StatePublished - 1995
Externally publishedYes

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4-Aminobenzoic Acid
Glutamine
Amination
Escherichia coli
Dithiothreitol
Folic Acid
Diazooxonorleucine
para-Aminobenzoates
Pyruvic Acid
Ammonia
Vitamins
Cysteine
Glutamic Acid
aminodeoxychorismate synthase

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli. / Viswanathan, Virinchipuram; Green, J. M.; Nichols, B. P.

In: Journal of Bacteriology, Vol. 177, No. 20, 1995, p. 5918-5923.

Research output: Contribution to journalArticle

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