Kanaya S, Koyanagi T, Kanaya E
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan.
Biochem J. 1998 May 15;332 ( Pt 1)(Pt 1):75-80. doi: 10.1042/bj3320075.
An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaC gene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degreesC and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coli esterase.
对大肠杆菌中一种属于激素敏感性脂肪酶（HSL）家族的酯酶进行了过量表达、纯化及特性鉴定。它由ybaC基因编码，由319个氨基酸残基组成，相对分子质量为36038。在25℃和pH 7.1条件下，以各种脂肪酸对硝基苯酯为底物测定酶活性。该酶对酰基链长度小于8的底物表现出水解活性，而对酰基链长度大于10的底物水解活性很小。此外，它对三油酰甘油和胆固醇油酸酯的水解活性也很小。对C2至C8底物水解动力学参数的测定表明，C4和C5底物是最适宜的。SDS/PAGE测定的相对分子质量（37000）与柱色谱法测定的结果（38000）相近，表明该酶以单体形式存在。它是一种酸性蛋白，pI值为4.1。远紫外圆二色光谱表明其螺旋含量为26.1%。将该酶的氨基酸序列与HSL家族其他成员的序列进行比较，我们推测Ser165、Asp262和His292构成了大肠杆菌酯酶的催化三联体。
