Valine, not methionine, is amino acid 106 in human cytosolic thymidine kinase (TK1). Impact on oligomerization, stability, and kinetic properties.
Cytosolic thymidine kinase (TK1) cDNA from human lymphocytes was cloned, expressed in Escherichia coli, purified, and characterized with respect to the ATP effect on thymidine affinity and oligomerization. Sequence analysis of this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from healthy cells or leukemic or transformed cell lines revealed a valine at amino acid position 106. The TK1 sequence in NCBI GenBank(TM) has methionine at this position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) has the same enzymatic and oligomerization properties as endogenous human lymphocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependent reversible transition from a dimer to a tetramer with 20-30-fold higher thymidine affinity (K(m) about 15 and 0.5 microm, respectively). Substitution of Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent tetramer with the high thymidine affinity (K(m) about 0.5 microm), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable than rLy-TK1(Val-106) (t(12) at 15 degrees 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.
Department of Life Sciences and Chemistry, Roskilde University, DK 4000 Roskilde, Denmark.
D
D
Berenstein
J F
JF
Christensen
T
T
Kristensen
R
R
Hofbauer
B
B
Munch-Petersen
D000255:Adenosine Triphosphate / Q000494:pharmacology; D019943:Amino Acid Substitution; D002461:Cell Line, Transformed; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D016208:Databases, Factual; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D008715:Methionine / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013936:Thymidine / Q000378:metabolism / Q000494:pharmacology; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014633:Valine / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008715:Methionine; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D014633:Valine; D013936:Thymidine