Schroeder William A, Locke Troy R, Jensen Susan E
Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Antimicrob Agents Chemother. 2002 Nov;46(11):3568-73. doi: 10.1128/AAC.46.11.3568-3573.2002.
In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a beta-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM beta-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP.
为了比较对克拉维酸抑制的耐药模式与对β-内酰胺酶抑制蛋白（BLIP）抑制的耐药模式，通过定点诱变制备了TEMβ-内酰胺酶的R164S、R244S和R164S/R244S突变形式。当测定这些TEM突变型和野生型的动力学参数时，单突变体表现出与文献中相似的特性，但双突变体表现出非常不同的特性。TEM的R164S/R244S双突变体形式保留了对克拉维酸抑制的耐药性（R244S突变的特征），但失去了所有水解头孢他啶的能力（R164S突变的特征）。虽然这些特征与先前报道的R164S/R244S双突变体的特征相反，但这种差异是由于早期研究中使用了有缺陷的突变体。当测试TEM的单突变体和双突变体形式对BLIP的抑制时，它们仍然高度敏感，与野生型相比仅显示出略有增加的耐药性；这种耐药模式与克拉维酸耐药模式有很大不同。在突变体中观察到的对BLIP抑制的耐药性略有增加可能与以下事实有关：所有突变都导致TEM蛋白净电荷发生变化，这可能会阻碍与BLIP的相互作用。
