(t1 ⁄2 at 15°C is 41 and 392 min, respectively). Because valine with high probability is the naturally occurring amino acid at position 106 in human TK1 and because this position has high impact on the enzyme properties, the Val-106 form should be used in future investigations of recombinant human TK1.The human cytosolic thymidine kinase, TK1 (EC 2.7.1.21), is a key enzyme in the salvage synthesis of TMP from thymidine. Intracellular TMP is quickly phosphorylated to TDP and TTP. Because TTP is an allosteric effector of ribonucleotide reductase, imbalances in the TTP pool disturb the supply of both purines and pyrimidines for DNA synthesis and repair. In turn, imbalanced deoxynucleoside triphosphate (dNTP) pools increase the mutation rate and probability of carcinogenesis (1-3).TK1 is a cell cycle-regulated enzyme. Its activity fluctuates with DNA synthesis, being high in dividing and malignant cells and low in quiescent cells (4, 5). The expression of TK1 is meticulously controlled on the transcriptional and post-transcriptional level (6, 7). At the enzymatic level, ATP, besides being a cosubstrate, has been shown to be a regulator of the catalytic activity of TK1 (8). Thus, exposure to ATP induces a reversible enzyme concentration-dependent transition from a low thymidine affinity dimer of about 50 kDa (K m ϭ 15 M) to a high affinity tetramer (K m ϭ 0.7 M). To further investigate the effect of ATP, we constructed a pET3a-TK1 plasmid (9) containing the amino acid coding region of TK1 cDNA from the pTK11 plasmid of Bradshaw and Deininger (10), who had used SV40 transformed human fibroblasts as the mRNA source. We expressed the resulting recombinant TK1 (rTFi-TK1) in Escherichia coli, and purified and characterized the enzyme. To our surprise we found that the enzymatic properties of rTFi-TK1 differed markedly from those of the endogenous Ly-TK1 with respect to the regulatory effect of ATP (9). Irrespective of preexposure to ATP, the recombinant rTFi-TK1 was a permanent tetramer of about 100 kDa with high affinity to thymidine with a K m value about 0.4 M (9). At that time, we assumed that the amino acid sequences of rTFi-TK1 and Ly-TK1 were identical and explained the divergent properties of rTFi-TK1 by the absence of post-translational modification of TK1 when expressed in E. coli (9). Because the pET3a-TK1 expression system was not satisfactory in terms of amount of TK1 protein produced, we constructed another expression plasmid, pGEX-2T-LyTK1. Here, the amino acid coding region of TK1 from normal human lymphocytes was cloned into the pGEX-2T vector, and the recombinant TK1 was expressed as an isopropyl-1-thio-␤-D-galactopyranoside-inducible glutathione S-transferase fusion protein. In contrast to the findings with rTFi-TK1, our preliminary kinetic experiments showed that the recombinant lymphocyte TK1 (rLy-TK1) 1 behaved essentially as the endogenous lymphocyte enzyme, Ly-TK1, regarding kinetic and oligomerization properties. Therefore, absence of posttranslational modification of rTFi-TK1 in E. coli cannot ex...