THYMIDINE KINASE MUTANTS OBTAINED BY RANDOM SEQUENCE SELECTION

Knowledge of the catalytic properties and structural information regar ding the amino acid residues that comprise the active site of an enzym e allows one, in principle, to use site-specific mutagenesis to constr uct genes that encode enzymes with altered functions. However, such in formation 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 v ariants. We describe here the construction, selection, and characteriz ation of herpes simplex virus type 1 thymidine kinase mutants either w ith different catalytic properties or with enhanced thermostability. F rom a library containing 2 x 10(6) plasmid-encoded herpes thymidine ki nase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this l ibrary and one previously constructed, we identified by secondary sele ction Escherichia coli harboring thymidine kinase mutant clones that w ere unable to grow in the presence of concentrations of 3'-azido-3'-de oxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced K (m) for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identifi ed one mutant with enhanced thermostability. These mutants may have cl inical potential as the promise of gene therapy is increasingly becomi ng a reality.