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, ex pressed in Escherichia coli, purified, and characterized with respect to th e ATP effect on thymidine affinity and oligomerization, Sequence analysis o f this lymphocyte TK1 cDNA and 21 other cDNAs or genomic TK1 DNAs from heal thy 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 t his position. The recombinant lymphocyte TK1(Val-106) (rLy-TK1(Val-106)) ha s the same enzymatic and oligomerization properties as endogenous human lym phocyte TK1 (Ly-TK1); ATP exposure induces an enzyme concentration-dependen t reversible transition from a dimer to a tetramer with 20-30-fold higher t hymidine affinity (K-m about 15 and 0.5 mu M, respectively). Substitution o f Val-106 with methionine to give rLy-TK1(Met-106) results in a permanent t etramer with the high thymidine affinity (K-m about 0.5 mu M), even without ATP exposure. Furthermore, rLy-TK1(Met-106) is considerably less stable th an rLy-TK1(Val-106) (t(1/2) at 15 degrees C is 41 and 392 min, respectively ). Because valine with high probability is the naturally occurring amino ac id at position 106 in human TK1 and because this position has high impact o n the enzyme properties, the Val-106 form should be used in future investig ations of recombinant human TK1.