6-Aminohexanoic acid cyclic dimer hydrolase. A new cyclic amide hydrolase produced by Achromobacter guttatus KI74.
6-Aminohexanoic acid cyclic dimer hydrolase. A new cyclic amide hydrolase produced by Achromobacter guttatus KI74.
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6-氨基己酸环状二聚体水解酶。
DOI：
10.1111/j.1432-1033.1977.tb11904.x
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发表时间：
1977
期刊：
影响因子：
--
通讯作者：
H. Okada
中科院分区：
文献类型：
--
作者：
S. Kinoshita;S. Negoro;M. Muramatsu;V. Bisaria;S. Sawada;H. Okada
以6-氨基己酸环二聚体(1，8-二氮杂环十四烷-2，9-二酮)为底物的滴状无色杆菌细胞产生的6-氨基己酸环二聚体水解酶，经聚丙烯酰胺圆盘凝胶电泳、十二烷基硫酸钠聚丙烯酰胺凝胶电泳法、Sephadex G-200柱层析、分析离心法纯化至均一。纯化后的酶具有以下特性。
(1)Sephadex G-200柱层析测得该酶的相对分子质量为110 000，沉淀平衡法测得该酶的相对分子质量为100 000，十二烷基硫酸钠聚丙烯酰胺凝胶电泳法测得该酶的最小相对分子质量为55 000，表明该酶为二聚体。由沉降速度推算出的沉降常数(S_(20))为5.7 S。
(2)以6-氨基己酸环二聚体为底物时，该酶的最适pH为7.3。该酶在pH为5.5~8.5范围内稳定，在45℃和50℃加热10min后，活性分别丧失50%和100%。最适反应温度为33℃，米氏常数(Km)为6 mm，周转数为8 S−1，相对分子质量为10万。
(3)该酶可被1μM二异丙基磷酸盐和10μM对氯汞苯甲酸强烈抑制，但几乎不被10 0 mM乙二胺四乙酸抑制。被对氯苯甲酸汞抑制的酶可被2-巯基乙醇重新激活。
(4)该酶仅对6-氨基己酸环状二聚体具有催化活性，可生成6-氨基己酰-6-氨基己酸。对6-氨基己酸低聚物(聚合度2~6)、各种环酰胺、环二酰胺、酰胺、寡肽和酪蛋白不起作用。
(5)酶的吸收系数(A1%280 nm)为19.4。该酶由500个氨基酸残基组成，其中包括3个半胱氨酸，由此计算出其二聚体的相对分子质量为103 200。
(6)6-氨基己酸环二聚体水解酶是一种新的环酰胺水解酶。
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.