肺炎克雷伯氏菌AmpC β-内酰胺酶动力学及抑酶效应的研究

Study of kinetics of AmpC β-lactamase in Klebsiella pneumoniae and the inhibitory activity of β-lactamase inhibitors

对肺炎克雷伯氏菌 99- 799株产生的 Amp C型β-内酰胺酶利用分光光度法进行了酶动力学及酶抑制剂抑酶效应的研究。分别以 L - B作图法求取米氏常数 (Km)和最大反应速度 (Vmax) ,以 Dixon作图法求取抑制常数 Ki,以半对数作图法求取酶活性被抑制 5 0 %时的抑制剂浓度 IC50 。结果表明 ,Amp C酶对头孢噻啶、头孢噻吩和头孢哌酮的 Km 及 Vmax/ Km 值较苯唑西林、头孢噻肟、头孢他啶、头孢西丁和氨曲南的值大 ,说明Amp C酶对头孢噻啶、头孢噻吩和头孢哌酮具有较低的亲和力和较高的水解效率。此外 ,克拉维酸、舒巴坦和三唑巴坦 3种β-内酰胺酶抑制剂对 Amp C酶的抑制效力均很差 ,尤其是克拉维酸作用很弱。

The enzyme kinetics of AmpC β-lactamase in Klebsiella pneumoniae 99-799 and the inhibitory activities of β-lactamase inhibitors were studied by spectrophotometry. Michaeles constant (K m) and maximal velocity (V max ) were obtained by L-B plot. The inhibition constant (K i) and 50% inhibitory concentration (IC 50 ) were analyzed by Dixon plot and the semilogarithmic plots, respectively. Results showed that the K m and V max / K m b values of cephaloridine (CER), cephalothin (CET) and cefoperazone (CPZ) were higher than those of oxacillin (OXA), cefotaxime (CTX), ceftazidime (CAZ), cefoxitin (CFX) and aztreonam (AZ). These findings suggested that AmpC β-lactamase had relatively lower affinity and higher efficiency of hydrolysis to CER, CET and CPZ. Furthermore, all the three β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) used in this study had poor effects against AmpC produced by K.pneumoniae 99-799, and clavulanic acid was the worst among them.