KINETIC CHARACTERIZATION OF 4-AMINO 4-DEOXYCBORISMATE SYNTHASE FROM ESCHERICHIA-COLI

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli i s its incorporation into the vitamin folic acid, PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeox ychorismate (ADC) synthase converts chorismate and glutamine to ADC an d glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA, Wh ile there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We repor t that PabA requires a preincubation with dithiothreitol for maximal a ctivity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB undergoes inactivation upon incu bation at 37 degrees C, which is prevented by the presence of chorisma te or PabA; glutamine enhances the protective effect of PabA, Incubati on with fresh dithiothreitol reverses the inactivation of PabB, We con clude that both PabA and PabB have cysteine residues which are essenti al for catalytic function and/or for subunit interaction, Using condit ions established for maximal activity of the proteins, we measured the K-m values for the glutamine-dependent and ammonia-dependent aminatio ns of chorismate, catalyzed by PabB alone and by the ADC synthase comp lex, Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L -norleucine were consistent with an ordered bi-bi mechanism in which c horismate binds first. No inhibition of ADC synthase activity was obse rved when p-aminobenzoate, sulfanilamide, sulfathiazole, and several c ompounds requiring folate for their biosynthesis were used.