title: Arg-188 and Trp-144 are implicated in the binding of urea and acetamide to the active site of the amidase from Pseudomonas aeruginosa.
abstract: Urea is a time-dependent active-site-directed inhibitor of Pseudomonas aeruginosa amidase. We found that 20 mM hydroxylamine caused bound urea to be released from the inactive urea:amidase complex with the restoration of enzyme activity. Bound urea restricts the titrability of the enzyme's -SH groups to 6 per hexameric molecule and protects it against thermal denaturation suggesting that urea binding provokes a conformational change in the enzyme. Mutations in the P. aeruginosa amidase gene that reduce the binding affinity of the enzyme for both urea and the substrate acetamide have been identified by direct sequencing of PCR-amplified mutant genes and confirmed by sequencing cloned PCR-amplified genes. The mutations were in two regions of the enzyme substituting either Arg-188 (or Gln-190, in one case) or Trp-144; one amidase that bound neither urea nor acetamide was doubly mutant with an amino-acid change at both sites.
authors.affiliation: Molecular Biology and Biophysics Section, King's College London, Strand, UK.
authors.forename: R
authors.initials: R
authors.lastname: Tata
authors.forename: P
authors.initials: P
authors.lastname: Marsh
authors.forename: P R
authors.initials: PR
authors.lastname: Brown
mesh_terms: D000081:Acetamides / Q000378:metabolism*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan; D014508:Urea / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000081:Acetamides; D014364:Tryptophan; D014508:Urea; C030686:acetamide; D001120:Arginine; D004228:Dithionitrobenzoic Acid; D000581:Amidohydrolases; C019665:amidase