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

Abstract

BackgroundDNA 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.ResultsThe 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.ConclusionsSteady-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.

Kinetic mechanism and fidelity of nick sealing byEscherichia coliNAD+-dependent DNA ligase (LigA)

“…To our knowledge, this is the first transient state kinetic analysis of a LigA–AMP enzyme. Our finding adds to existing steady-state kinetic studies of the NAD + -dependent nick joining reactions of Eco LigA and Haemophilus influenzae LigA, both of which are stimulated strongly by ammonium sulfate ( 37 , 52 , 53 ). We found that ammonium ion enhances the rate of step 2 and step 3 catalysis by Eco LigA–AMP, as much as 53-fold and 20-fold, respectively, at 15 mM ammonium sulfate.…”

The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

“…[ 36 ] In all cases, the inhibition equilibrium constant (K i ) of the 75mer-ds-nDNA substrate was estimated to be 200–500 nM for these ligases. In addition to uncompetitive substrate inhibition, the inhibition could also potentially be explained by a competitive model, with nicked substrate binding to deadenylylated ligase inhibiting the self-adenylylation reaction through blocking binding of ATP [ 6 , 37 ]. Thus, the T4 substrate inhibition data was also fit with a competitive substrate inhibition model for an ordered Bi-Bi Ping-Pong mechanism ( Eq 3 ).…”

“…[ 6 , 37 ] For these enzymes, it was hypothesized that the underlying mechanistic reason for the inhibition was the competitive binding of deadenylylated enzyme to the nicked substrate, thus inhibiting enzyme self-adenylylation. [ 6 , 37 ] In this study, EMSAs performed with a deadenylylated form of T4 DNA ligase clearly demonstrated that stable binding of nicked DNA does occur for this ligase ( Fig 4 ), providing direct evidence for potential inhibition of enzyme self-adenylylation through this mechanism. An important corollary to this substrate inhibition mechanistic hypothesis is that the studies of H .…”

“…Thus, a large potential effect on ligase self-adenylylation rates might be expected. [ 6 , 37 ] By contrast, the T4 phage’s E . coli host has a free intracellular ATP concentration of 1.5 mM ± 1.2 mM, [ 43 ] comparable to the 1 mM ATP concentration used in this study.…”

Biochemical and structural characterization of DNA ligases from bacteria and archaea

“…A wide range of biophysical chemistry approaches have been used to detect the joining of DNA strand breaks. Those applied to NDLs or ADLs from bacteria or archaea include use of fluorescently-labelled probes [ 66 , 70 , 72 , 73 ], FRET [ 69 , 74 – 77 ], fluorescence quenching and molecular beacon-based approaches [ 78 – 81 ], electrochemical methods [ 67 , 79 , 81 – 84 ], a nanoparticle-based sensor [ 85 ] and surface plasmon resonance [ 86 ]. Biochemical analyses of the ligation reaction are facilitated by assays that do not require ‘labelling’ of the nucleic acid, such as label-free electrochemical approaches using mercury-based electrodes and nicked plasmid DNA substrates [ 67 ].…”