344 Maunders
The bacteriophage polynucleotide kinases have similar properties,
that from T4-infected E. coli having been the most studied. In this
chapter, I concentrate on T4 polynucleotide kinase as an example of
these enzymes.
- The Enzyme
T4 polynucleotide kinase is encoded by the structural gene pseT.
This gene also codes for a T4 3'-phosphatase (8-10), whose activity
has been identified as residing on the same enzyme molecule. The mutant
phage pseT1 lacks the phosphatase activity (11-13), but has unaffected
kinase activity, and is therefore often used as a source for preparing the
enzyme. Like many commercially available enzymes, polynucleotide
kinase has also been prepared from a recombinant overproducing strain.
T4 polynucleotide kinase is a tetramer composed of identical subunits,
each consisting of 45-55% t~-helix and possessing an N-terminal pheny-
lalanine residue (14). The mol mass of the native enzyme has been deter-
mined as 140,000 Daby gel filtration (15), and 147,300 Daby centrifugation
(14). The size of the denatured and reduced monomers has been measured
as 33,000 Da by polyacrylamide gel electrophoresis (14,15), and 33,200
Da by centrifugation (14). The sedimentation coefficients, S°20,w, are 2.95 S
and 6.55S for the monomer and tetramer respectively.
By comparison, the rat liver enzyme has been determined to have a
mol mass of 80,000 Da by gel filtration (16,17), and a sedimentation
coefficient of 4.4S (16).
- Enzymic Reaction
3.1. Reaction Catalyzed
T4 polynucleotide kinase catalyzes the transfer of the T-phosphate
group from a 5'-nucleoside triphosphate to the 5'-OH of an acceptor
molecule. This may be a nucleoside-3'-phosphate, an oligonucleotide,
or a polynucleotide. The reaction is reversible, and the enzyme will
catalyze polynucleotide dephosphorylation in the presence ofa nucleo-
tide diphosphate such as ADP (18). Excess ADP will cause the reverse
reaction to be favored.
The reverse reaction can be utilized for the exchange of labeled
phosphate groups between the two substrates. This exchange reaction
allows 5'-labeling of polynucleotides without prior removal of the
existing 5'-phosphate group.
