Venkatachalam K V, Huang W, LaRocco M, Palzkill T
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1994 Sep 23;269(38):23444-50.
Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238S:E240K double mutant. Each enzyme was purified to homogeneity and the kinetic parameters kcat and Km were determined using several substrates. The G238S substitution increases catalytic efficiency for both ceftazidime and cefotaxime. However, to achieve large increases in catalytic efficiency, both G238S and the E240K substitutions are required. The R241G substitution results in a small increase in catalytic efficiency for only ceftazidime. The contribution of each residue to the transition-state stabilization energy was found to be additive indicating that the substitutions act independently to change the catalytic properties of the enzyme.
最近，在对头孢噻肟和头孢他啶等超广谱头孢菌素耐药性增强的天然分离株中，已鉴定出在TEMβ-内酰胺酶活性位点口袋中有氨基酸取代的变体。为了鉴定其他改变TEM-1对超广谱头孢菌素活性的氨基酸取代，构建了一个随机文库，其中包含238-241（ABL编号）3个残基窗口内的所有可能氨基酸取代。选择对头孢他啶的耐药性比野生型高100倍的突变体。所有突变体在238位有丝氨酸取代，在240位有赖氨酸或精氨酸，在241位有小氨基酸。通过构建单个G238S、E240K和R241G取代以及G238S:E240K双突变体来研究每个取代的作用。将每种酶纯化至同质，并使用几种底物测定动力学参数kcat和Km。G238S取代提高了对头孢他啶和头孢噻肟的催化效率。然而，为了实现催化效率的大幅提高，G238S和E240K取代都需要。R241G取代仅导致对头孢他啶的催化效率略有提高。发现每个残基对过渡态稳定能的贡献是累加的，这表明这些取代独立作用以改变酶的催化特性。
