Regulation of the activity and polymerization status of recombinant human cytosolic thymidine kinase by thiols and ATP

The cDNA clone encoding human thymidine kinase (hTK), was expressed in E. c oli using a prokaryotic expression vector, pKK 223-3. The kinetics of the r ecombinant hTK (rhTK) were similar to those of cytosolic TK but not of mito chondrial TK. rhTK was highly purified in the presence of either ATP or dit hiothreitol (DTT). The specific activity of rhTK purified in the presence o f ATP [rhTK(ATP)] was lower than that of rhTK purified in the presence of D TT [rhTK(DTT)]. Activity of the purified rhTK(ATP) was enhanced by addition of thiols including DTT, cysteine, homocysteine and beta-mercaptoethanol b ut inhibited by various sulfhydryl reagents such as 5,5'-dithio-bis(2-nitro benzoic acid). Hence, it was suggested that rhTK is a thiol-type enzyme. Ap parent Mr of purified rhTK(ATP) was 100 kDa, which corresponds to the size of a tetramer (25 kDa subunit), while that of purified rhTK(DTT) was 50 kDa , the size of;a dimer. The tetramer form of rhTK(ATP) was converted to the dimer by replacement of ATP by DTT. On the other hand, the dimer form of rh TK(DTT) was converted to the tetramer by addition of ATP. Thus, the catalyt ic activity of human cytosolic TR might be regulated by thiols as well as A TP via its polymerization status.