Lee Y L, Chen J C, Shaw J F
Department of Marine Food Science, National Taiwan Ocean University, Keelung.
Biochem Biophys Res Commun. 1997 Feb 13;231(2):452-6. doi: 10.1006/bbrc.1997.5797.
A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109. The overexpressed, matured enzyme from JM109 was purified to homogeneity. The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives. The enzyme had a kcat/Km value of 0.363 s-1 microM-1, for a typical thioesterase I substrate, palmitoyl-CoA. The arylesterase activity of the enzyme was observed by its ability to hydrolyze several aromatic esters including alpha-naphthyl acetate, alpha-naphthyl butyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters. In kinetic studies a chymotrypsin-like substrate (an amino acid derivative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE), was the best substrate for the enzyme with a catalytic efficiency (kcat/Km) of 4.00 s-1 microM-1, which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s-1 microM-1). It was concluded that the thioesterase I of E. coli had arylesterase activity and it possessed stereospecificity for protease substrates.
从大肠杆菌JM109菌株中重新克隆并测序了硫酯酶I基因。对来自JM109的过表达、成熟的酶进行了纯化，使其达到同质。该酶对包括酰基辅酶A、酯和氨基酸衍生物在内的三种底物表现出广泛的水解活性。对于典型的硫酯酶I底物棕榈酰辅酶A，该酶的kcat/Km值为0.363 s-1 microM-1。通过其水解几种芳香酯（包括乙酸α-萘酯、丁酸α-萘酯、乙酸苯酯、乙酸苄酯和八种对硝基苯酯）的能力观察到该酶的芳基酯酶活性。在动力学研究中，一种类胰凝乳蛋白酶底物（一种氨基酸衍生物），N-苄氧羰基-L-苯丙氨酸对硝基苯酯（L-NBPNPE）是该酶的最佳底物，催化效率（kcat/Km）为4.00 s-1 microM-1，比其对映体D-NBPNPE（0.171 s-1 microM-1）高23倍。得出结论，大肠杆菌的硫酯酶I具有芳基酯酶活性，并且对蛋白酶底物具有立体特异性。
