Characterization of an Inducible Amidase from Pseudomonas acidovorans AE 1

Abstract

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 57500 as determined by gel filtration and 55300 as computed from the amino acid composition. By polyacrylamide gel electrophoresis in dodecylsulfate a polypeptide chain weight of 56700 was obtained. Based on the reaction of the highly purified enzyme with diethyl‐4‐nitrophenyl phosphate an equivalent weight of approximately 59100 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 HgCl2 or 4‐chloromercuribenzoic acid. o‐Nitrophenol caused a competitive inhibition and phenetidine an uncompetitive inhibition.

Purification and characterization of aryl acylamidase from Nocardia globerula

“…Aryl acylamidase has been purified from monkey brain [17], human erythrocytes and liver [19] and rat serum [18], or to homogeneity from human serum [18] and sheep platelets [20], and their relationship to acetylcholinesterase in the corresponding tissue has been investigated. The enzyme has also been partially purified [25] [24]; P. acidovorans, 55-57 kDa [15]. Compared to them, our enzyme has much higher molecular m a s (126 kDa) and is composed of two subunits.…”

“…In addition, when the acetanilide derivatives have a substitution in the ortho position, the rate of reaction was decreased by steric hindrance. The hydrolysis of L-leucine-pnitroanilide catalyzed by Pseudomonas acidovorans aryl acylamidase has been reported [15]. The hydrolytic activity of our enzyme toward various L-amino-acid-p-nitroanilide derivatives was also investigated ( Table 5).…”

“…However, the hydrolysis of acetamide was barely detected even when 10-fold higher enzyme concentrations were added and The enzyme also catalyzed the hydrolysis of esters such as phenyl acetate and methyl benzoate at a considerable rate. The hydrolysis of phenylacetate, 4-nitrophenyl acetate and 2-nitrophenyl acetate catalyzed by P. acidovorans aryl acylamidase has been reported [15].…”

Isolation of an Inducible Amidase from Pseudomonas acidovorans AE1

“…The enzyme was strongly inhibited by diethyl-p-nitrophenyl phosphate (Alt, Heymann & Krisch, 1975). The highest specific activity, which was achieved in three out of eight preparations, was 137 pmol/min/mg.…”

“…The highest specific activity, which was achieved in three out of eight preparations, was 137 pmol/min/mg. Only with these preparations was the equivalent weight identical with the molecular weight as determined by gel filtration and with the chain weight as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (Alt et al 1975).…”