Schultz S C, Richards J H
Proc Natl Acad Sci U S A. 1986 Mar;83(6):1588-92. doi: 10.1073/pnas.83.6.1588.
A mutagenic technique that "saturates" a particular site in a protein with all possible amino acid substitutions was used to study the role of residue 71 in beta-lactamase (EC 3.5.2.6). Threonine is conserved at residue 71 in all class A beta-lactamases and is adjacent to the active site Ser-70. All 19 mutants of the enzyme were characterized by the penam and cephem antibiotic resistance they provided to Escherichia coli LS1 cells. Surprisingly, cells producing any of 14 of the mutant beta-lactamases displayed appreciable resistance to ampicillin; only cells with mutants having Tyr, Trp, Asp, Lys, or Arg at residue 71 had no observable resistance to ampicillin. However, the mutants are less stable to cellular proteases than wild-type enzyme is. These results suggest that Thr-71 is not essential for binding or catalysis but is important for stability of the beta-lactamase protein. An apparent change in specificity indicates that residue 71 influences the region of the protein that accommodates the side chain attached to the beta-lactam ring of the substrate.
一种诱变技术被用于研究β-内酰胺酶（EC 3.5.2.6）中71位残基的作用，该技术用所有可能的氨基酸替换使蛋白质中的特定位点“饱和”。在所有A类β-内酰胺酶中，71位残基的苏氨酸是保守的，且与活性位点Ser-70相邻。通过它们赋予大肠杆菌LS1细胞的青霉烯和头孢烯抗生素抗性对该酶的所有19种突变体进行了表征。令人惊讶的是，产生14种突变β-内酰胺酶中任何一种的细胞对氨苄青霉素都表现出明显的抗性；只有71位残基为酪氨酸、色氨酸、天冬氨酸、赖氨酸或精氨酸的突变体的细胞对氨苄青霉素没有明显抗性。然而，这些突变体比野生型酶对细胞蛋白酶的稳定性更低。这些结果表明，Thr-71对于结合或催化不是必需的，但对β-内酰胺酶蛋白的稳定性很重要。特异性的明显变化表明，71位残基影响蛋白质中容纳与底物β-内酰胺环相连的侧链的区域。
