346 MaundersThe following K m values have been determined for T2 polynucleo-
tide kinase: 14 lxM for ATP, 15 ~$/for UTP, 33 lxM for GTP, and 25 JaM
for CTP (21). With the T4 enzyme, the K m value for ATP is 13-140 JaM
depending on the DNA acceptor (3,24).
Under the conditions for phosphate exchange, the KmATP (forward
reaction) is 4 ~$/, and the KmADP (reverse reaction) is 200 ~/. The
optimal concentrations for exchange are 10 ~/ATP and 300 laMADP
(20), but even so reaction is usually incomplete.
For the dephosphorylation of single-stranded oligonucleotides,
KmADP is 0.22 laM (18), but again only partial dephosphorylation can
be achieved.
3.3. Temperature
As with most E. coli bacteriophage-derived enzymes, the optimal
reaction temperature is 37°C.
T4 polynucleotide kinase will also perform at lower temperatures,
which is a useful attribute. The rate of the kinase reaction at 0°C is
reduced to 7% of that at 37°C. However, the exchange reaction rate is
reduced much further, to 1.2% of that at 37°C (25), so under these
conditions, kinasing is greatly favored.
3.4. pH
The optimum pH range for T4 polynucleotide kinase is 7.4-8.0, with
maximum activity in Tris buffers being observed at pH 7.6 (2). The
reverse reaction has a pH optimum of 6.2, in imidazolebuffer (20).
The 3'-phosphatase activity is greatest at pH 5.9 (9).
3.5. Cations
Polynucleotide kinase has a requirement for magnesium ions for
both the forward and exchange reactions (18), with no activity being
detectable in their absence. At the optimal pH of 7.6, the optimum
magnesium concentration is 10 mM (2).
Manganese can partially replace magnesium in some cases. Note
that 3.3 mM Mn 2÷ will permit reaction at 50% of the maximum rate
obtainable with 10 mM Mg 2÷.
The 3'-phosphatase activity similarly requires Mg 2÷, with the reac-
tion rate falling to 2% in its absence. In this case Co 2÷ can replace Mg 2÷
to some extent (21).