Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli.
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 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 Km 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.
Viswanathan
V K
VK
Department of Biological Sciences, University of Illinois at Chicago 60607-7060, USA.
Green
J M
JM
Nichols
B P
BP
D010129:4-Aminobenzoic Acid / Q000494:pharmacology; D000586:Amination; D000641:Ammonia / Q000494:pharmacology; D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism*; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005246:Feedback; D005973:Glutamine / Q000494:pharmacology; D007700:Kinetics; D000637:Transaminases / Q000187:drug effects / Q000378:metabolism*; D062366:para-Aminobenzoates
D001426:Bacterial Proteins; D004793:Enzyme Reactivators; D029968:Escherichia coli Proteins; D062366:para-Aminobenzoates; D005973:Glutamine; D000641:Ammonia; D000637:Transaminases; C000707653:aminodeoxychorismate synthase protein, E coli; C066599:aminodeoxychorismate synthase; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid; D004229:Dithiothreitol; D010129:4-Aminobenzoic Acid