Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2.
A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.
Shimizu
S
S
Department of Agricultural Chemistry, Kyoto University, Japan.
Ogawa
J
J
Chung
M C
MC
Yamada
H
H
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010627:Phenethylamines / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013696:Temperature
D000596:Amino Acids; D002413:Cations, Divalent; D046911:Macromolecular Substances; D010627:Phenethylamines; D013438:Sulfhydryl Compounds; C054480:N-acetyl-2-phenylethylamine; D000581:Amidohydrolases; C077287:arylalkyl acylamidase