Lin S, Thomas M, Shlaes D M, Rudin S D, Knox J R, Anderson V, Bonomo R A
Research Service, Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH 44105, USA.
Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):395-400. doi: 10.1042/bj3330395.
The Met69-->Ile mutant of the OHIO-1 beta-lactamase, an SHV-family enzyme, is resistant to inactivation by beta-lactamase inhibitors. Analysis of purified Met69-->Ile enzyme reveals that its isoelectric point (pI 7.0) and CD spectrum are identical with those of the OHIO-1 enzyme. Levels of beta-lactamase expression in Escherichia coli as determined by immunoblotting are similar for OHIO-1 and Met69-->Ile beta-lactamase. The kinetic constants of the Met69-->Ile enzyme compared with OHIO-1 are smaller for benzylpenicillin (Km = 6 microM compared with 17 microM; kcat = 234 s-1 compared with 345 s-1 respectively) and carbenicillin (Km = 3 microM compared with 17 microM; kcat = 131 s-1 compared with 320 s-1 respectively). For the cephalosporins cephaloridine and 7-(thienyl- 2-acetamido)-3-[2-(4-N,N- dimethylaminophenylazo)pyridinium-methyl]-3-cephem-4-carboxylic acid (PADAC), a similar pattern is also seen (Km=38 microM compared with 96 microM and 6 microM compared with 75 microM respectively; kcat = 235 s-1 compared with 1023 s-1 and 9 s-1 compared with 50 s-1 respectively). Consistent with minimum inhibitory concentrations that show resistance to beta-lactam beta-lactamase inhibitors, the apparent Ki values, turnover numbers and partition ratios (kcat/kinact) for the mechanism-based inactivators clavulanate, sulbactam and tazobactam are increased. The inactivation rate constants (kinact) are decreased. The difference in activation energy, a measurement of altered affinity for the wild-type and mutant enzymes leading to acylation of the active site, reveals small energy differences of less than 8.4 kJ/mol. In total, these results suggest that the Met-->Ile substitution at position 69 in the OHIO-1 beta-lactamase alters the active site, primarily affecting the interactions with beta-lactamase inhibitors.
OHIO-1β-内酰胺酶（一种SHV家族酶）的Met69→Ile突变体对β-内酰胺酶抑制剂的失活具有抗性。对纯化的Met69→Ile酶的分析表明，其等电点（pI 7.0）和圆二色光谱与OHIO-1酶相同。通过免疫印迹法测定，大肠杆菌中OHIO-1和Met69→Ileβ-内酰胺酶的β-内酰胺酶表达水平相似。与OHIO-1相比，Met69→Ile酶对苄青霉素的动力学常数较小（Km分别为6μM和17μM；kcat分别为234 s-1和345 s-1），对羧苄青霉素的动力学常数也较小（Km分别为3μM和17μM；kcat分别为131 s-1和320 s-1）。对于头孢菌素头孢菌素和7-（噻吩-2-乙酰氨基）-3-[2-（4-N，N-二甲基氨基苯基偶氮）吡啶甲基]-3-头孢烯-4-羧酸（PADAC），也观察到类似的模式（Km分别为38μM和96μM以及6μM和75μM；kcat分别为235 s-1和1023 s-1以及9 s-1和50 s-1）。与对β-内酰胺β-内酰胺酶抑制剂耐药的最低抑菌浓度一致，基于机制的灭活剂克拉维酸、舒巴坦和他唑巴坦的表观Ki值、周转数和分配比（kcat/kinact）增加。灭活速率常数（kinact）降低。活化能的差异（衡量野生型和突变酶对活性位点酰化的亲和力变化）显示出小于8.4 kJ/mol的小能量差异。总体而言，这些结果表明，OHIO-1β-内酰胺酶第69位的Met→Ile取代改变了活性位点，主要影响与β-内酰胺酶抑制剂的相互作用。
