PNK carries out two types of enzymatic activity:

•  Forward reaction: γ-phosphate is transferred from ATP to the 5' end of a polynucleotide (DNA or RNA). 5' phosphate is not present either due to chemical synthesis or dephosphorylation. The 5' OH nucleophile is activated by abstraction of the proton. Asp35 of PNK forms the co-ordinate bond with 5' OH and attacks γ phosphorus forming an intermediate.

•  Exchange reaction: target DNA or RNA having a 5' phosphate is incubated with an excess of ADP - where PNK transfers the phosphate from the nucleic acid to an ADP, forming ATP. PNK then performs a forward reaction and transfer a phosphate from ATP to the target nucleic acid. Exchange reaction is used to label with radioactive phosphate group.

Fig 2-4.2: Polynucleotide kinase reaction (A) forward (B) exchange.

(Adapted from http://www.vivo.colostate.edu/hbooks/genetics/biotech/)