Characterization of an inducible amidase from Pseudomonas acidovorans AE1.
The main molecular and catalytic properties of an acetanilide-hydrolyzing enzyme from Pseudomonas acidovorans AE 1, purified to a homogeneous state, were investigated. The molecular weight was 57 500 as determined by gel filtration and 55 300 as computed from the amino acid composition. By polyacrylamide gel electrophoresis in dodecylsulfate a polypeptide chain weight of 56 700 was obtained. Based on the reaction of the highly purufied enzyme with diethyl-4-nitrophenyl phosphate an equivalent weight of approximately 59 100 was found. From these results it was concluded that the enzyme consists of a single polypeptide chain and contains one active site per molecule. The enzyme hydrolyzed esters as well as certain aromatic amides. It also catalysed the transfer of acetyl groups to phenetidine yielding phenacetin. The activities towards aliphatic esters were much smaller. The enzyme was stable at pH values ranging from 7 to 9 and its pH-optimum was about 10. It was strongly inhibited by organophosphorous compounds, like diethyl-4-nitrophenyl phosphate or diisopropylphosphorofluoridate, as well as by physostigmine sulfate and -SH-blocking reagents, like HgCl-2 or 4-chloromercuribenzoic acid. o-Nitrophenol caused a competitive inhibition and phenetidine an uncompetitive inhibition.
D000083:Acetanilides / Q000031:analogs & derivatives; D000581:Amidohydrolases / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009574:Nitro Compounds; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D000083:Acetanilides; D000596:Amino Acids; D046911:Macromolecular Substances; D009574:Nitro Compounds; D009943:Organophosphorus Compounds; D000581:Amidohydrolases