p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.
p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.
Roux
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Walsh
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D010129:4-Aminobenzoic Acid / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005973:Glutamine; C022309:PABA synthase; D000637:Transaminases; C000707653:aminodeoxychorismate synthase protein, E coli; C066599:aminodeoxychorismate synthase; D005972:Glutaminase; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D002827:Chorismic Acid; D010129:4-Aminobenzoic Acid