p-Aminobenzoate synthesis in Escherichia coli: mutational analysis of three conserved amino acid residues of the amidotransferase PabA.
p-Aminobenzoate synthesis in Escherichia coli requires three enzymes, PabA, PabB, and PabC, acting respectively as glutaminase, chorismate aminase, and 4-amino-4-deoxychorismate aromatase. PabA requires stoichiometric amounts of PabB to display glutaminase activity. PabA has conserved cysteine (C79), histidine (H168), and glutamate (E170) residues that have been suggested in the analogous anthranilate synthase to form a type of catalytic triad in an acylenzyme mechanism. Mutations at each of these residues of PabA lead to the following observations. C79S PabA has 40-fold lower kcat and 10(4) lower kcat/Km with no detectable acylenzyme accumulation in steady-state turnover (vs wild-type PabA at 0.56 mol fraction of gamma-glutamyl-enzyme). H168Q has no catalytic activity and does not compete with wild-type PabA for PabB (this may indicate a folding defect). Four E170 mutants give three outcomes. E170D and E170A yield active PabA species, down 4-fold and 150-fold, respectively, in kcat/Km ratios from wild-type PabA. E170Q has no detectable glutaminase activity but does bind to PabB in competition with wild-typoe PabA while E170K has neither detectable catalytic activity nor the ability to be recognized by PabB.
Roux
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Walsh
C T
CT
D010129:4-Aminobenzoic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D000637:Transaminases / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D000637:Transaminases; C000707653:aminodeoxychorismate synthase protein, E coli; C066599:aminodeoxychorismate synthase; D019755:Carbon-Carbon Lyases; D019731:Carbon-Nitrogen Ligases; D010129:4-Aminobenzoic Acid