Munir K M, French D C, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Proc Natl Acad Sci U S A. 1993 May 1;90(9):4012-6. doi: 10.1073/pnas.90.9.4012.
Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.
了解构成酶活性位点的氨基酸残基的催化特性和结构信息，原则上可使人利用位点特异性诱变构建编码功能改变的酶的基因。然而，关于大多数酶的此类信息尚不清楚，特定氨基酸取代的效果通常也无法预测。另一种方法是在基因中用随机核苷酸取代关键密码子，并利用遗传筛选来鉴定新的、有趣的酶变体。我们在此描述了具有不同催化特性或增强热稳定性的1型单纯疱疹病毒胸苷激酶突变体的构建、筛选和特性分析。从一个包含2×10⁶个质粒编码的疱疹胸苷激酶基因的文库中，每个基因在假定的核苷结合位点具有不同的核苷酸序列，我们获得了1540个活性突变体。利用这个文库和一个先前构建的文库，我们通过二次筛选鉴定出携带胸苷激酶突变体克隆的大肠杆菌，这些克隆在存在允许携带野生型质粒的大肠杆菌形成菌落的3'-叠氮-3'-脱氧胸苷（AZT）浓度下无法生长。其中两个突变酶对AZT的Km值降低，其中一个相对于野生型对AZT的催化效率高于对胸苷的催化效率。我们还鉴定出一个具有增强热稳定性的突变体。随着基因治疗的前景日益成为现实，这些突变体可能具有临床应用潜力。
