Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection.
We have obtained 190 active Herpes simplex virus type 1 thymidine kinase mutants by substituting a 33 nucleotide sequence with 20% degeneracy for a portion of the nucleotide sequence that encodes the putative thymidine binding site [K.M. Munir, D.C. French, D.K. Dube and L.A. Loeb (1992) J. Biol. Chem., 167, 6584-6589]. In order to classify these mutants with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the presence of varying concentrations of thymidine. Escherichia coli harboring one of the mutant enzymes was able to form colonies at a concentration of thymidine lower than did the wild type. It was able to phosphorylate 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 [methyl-3H]thymidine into E. coli, (ii) a 2.4 times higher rate of [methyl-3H]thymidine incorporation into acid-insoluble material and (iii) a 5-fold increase in the kcat of the purified enzyme compared to the wild type. Herpes thymidine kinase purified from other mutants that formed colonies at higher thymidine concentrations than that of the wild type exhibited a decrease in kcat. The kcat of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme. This study demonstrates that a spectrum of mutant enzymes with different catalytic properties can be obtained by selection from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design.
Munir
K M
KM
Joseph Gottstein Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
French
D C
DC
Dube
D K
DK
Loeb
L A
LA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli; D006561:Herpes Simplex / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012641:Selection, Genetic; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D013936:Thymidine