Kinetic equation for template-dependent polymerase reactions and its application to Thermus thermophilus DNA polymerase

A kinetic equation was derived for complex DNA polymerase reactions. The co mplex repertoire of polymerase reactions includes the facts that the four m onomeric substrates involved in the polymerase reactions may also work as c ompetitive inhibitors depending on their incorporation sites, the incorpora tion rates of each deoxynucleotide are different, and polymerization procee ds in parallel at various sites on each template DNA. Using the equation, t he kinetic properties of Thermus thermophilus DNA polymerase were investiga ted. The reaction at the A-incorporation site is strongly inhibited by dTTP and dGTP with inhibition constants of 9.9 and 7.1 mu M, respectively. The reaction at the T-incorporation site is also inhibited by dATP with an inhi bition constant of 7.6 mu M, while the G- and C-incorporation sites are les s sensitive to competitive inhibition. When substrate concentrations are lo w, the incorporation rate of A is the fastest and that of C is the slowest. Accordingly, the C-incorporation step largely limits the overall reaction rate. Using the kinetic parameters determined experimentally, the optimum r atio of the concentrations of the four nucleotides was calculated.