Complete steady-state rate equation for DNA ligase and its use for measuring product kinetic parameters of NAD(+)-dependent DNA ligase from Haemophilus influenzae

BACKGROUND: DNA ligase seals the nicks in the phosphodiester backbone between Okazaki fragments during DNA replication. DNA ligase has an unusual Bi Ter Ping Pong kinetic mechanism. Its substrates in eubacteria are NAD(+) and nicked DNA (nDNA). Its products are nicotinamide mononucleotide (NMN), adenosine 5′-monophosphate (AMP), and sealed DNA. Investigation of the kinetic mechanism and measurement of the kinetic constants of DNA ligase using steady-state kinetics would benefit from the availability of the complete steady-state rate equation, including terms for product concentrations and product-related kinetic constants, which has not previously been published. RESULTS: The rate equations for two possible Bi Ter kinetic mechanisms for DNA ligase, including products, are reported. The mechanisms differ according to whether the last two products, AMP and sealed DNA, are released in an ordered or rapid-equilibrium random (RER) manner. Steady-state kinetic studies of product inhibition by NMN and AMP were performed with Haemophilus influenzae NAD(+)-dependent DNA ligase. The complete rate equation enabled measurement of dissociation constants for NAD(+), NMN, and AMP and eliminated one of 3 possible product release mechanisms. CONCLUSIONS: Steady-state kinetic product inhibition experiments and complete steady-state kinetic rate equations were used to measure dissociation constants of NAD(+), NMN, and AMP and eliminate the possibility that AMP is the second product released in an ordered mechanism. Determining by steady-state kinetics whether the release of sealed DNA and AMP products goes by an ordered (AMP last off) or RER mechanism was shown to require a product inhibition study using sealed DNA.