6‐Aminohexanoic Acid Cyclic Dimer Hydrolase. A New Cyclic Amide Hydrolase Produced by Acromobacter guttatus KI72

Abstract

6‐Aminohexanoic acid cyclic dimer hydrolase produced by Achromobacter guttatus cells grown on 6‐aminohexanoic acid cyclic dimer (1,8‐diazacyclotetradecane‐2,9‐dione) was purified until it was homogeneous according to polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, Sephadex G‐200 column chromatography, and analytical centrifugation. The following characteristics of the purified enzyme were observed.(1) The molecular weight of the enzyme, estimated to be 110 000 by column chromatography on Sephadex G‐200 and 100 000 by sedimentative equilibrium, together with the minimum molecular weight of 55 000 obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicated it is a dimeric enzyme. The sedimentation constant (s20) was estimated to be 5.7 S from the sedimentation velocity.(2) The enzyme had an optimum pH of 7.3 when 6‐aminohexanoic acid cyclic dimer was used as substrate. It was stable in a pH range between 5.5 to 8.5, and 50% and 100% of activity was lost after 10 min of heating at 45°C and 50°C respectively. The optimum temperature for activity was 33°C. The Michaelis‐Menten constant (Km) was 6 mM toward the dimer, and the turnover number was 8 s−1 assuming a molecular weight of 100 000.(3) This enzyme was strongly inhibited by 1 μM diisoprpylphosphofluoridate and 10 μM p‐chloromercuribenzoate but scarcely inhibited by 100 mM ethylenediaminetetraacetic acid. The enzyme inhibited by p‐chloromercuribenzoate could be reactivated by 2‐mercaptoethanol.(4) This enzyme was only active on 6‐aminohexanoic acid cyclic dimer to form 6‐aminohexanoyl‐6‐aminohexanoic acid. It was inactive on 6‐aminohexanoic acid oligomers (degree of polymerization 2 to 6), various cyclic amides, cyclic diamides, amides, oligopeptides, and casein.(5) The absorption coefficient (A1%280 nm) of the enzyme was 19.4. The enzyme was composed of 500 amino acid residues including 3 cysteines; from this the molecular weight was calculated to be 103 200 as a dimer.(6) 6‐Aminohexanoic acid cyclic dimer hydrolase was classified as a new cyclic amide hydrolase.