HERPES THYMIDINE KINASE MUTANTS WITH ALTERED CATALYTIC EFFICIENCIES OBTAINED BY RANDOM SEQUENCE SELECTION

We have obtained 190 active Herpes simplex virus type 1 thymidine kina se mutants by substituting a 33 nucleotide sequence with 20% degenerac y for a portion of the nucleotide sequence that encodes the putative t hymidine binding site [K.M.Munir, D.C.French, D.K.Dube and L.A.Loeb (1 992) J. Biol. Chem., 167, 6584-65891. In order to classify these mutan ts with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the p resence of varying concentrations of thymidine. Escherichia coli harbo ring one of the mutant enzymes was able to form colonies at a concentr ation of thymidine lower than did the wild type. It was able to phosph orylate thymidine more rapidly than the wild type both in vivo and in vitro. The increased thymidine kinase activity was manifested by (i) a 42% enhanced uptake of [methy 1-H-3]thymidine into E.coli, (ii) a 2.4 times higher rate of [methyl-H-3]thymidine incorporation into acid-in soluble material and (iii) a 5-fold increase in the k(cat) of the puri fied enzyme compared to the wild type. Herpes thymidine kinase purifie d from other mutants that formed colonies at higher thymidine concentr ations than that of the wild type exhibited a decrease in k(cat). The k(cat) of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme. This study demonstrates that a spectrum of mutan t enzymes with different catalytic properties can be obtained by selec tion from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design.