Abolfazl Arabshahi

Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159.

Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pK a of 8.4 ± 0.1. The simplest interpretation is that the apparent pK a is that of the 6-amino group of the AMP-accepting residue Lys159. Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0.0213 at 25 °C, corresponding to ΔG′o = +2.3 kcal mol−1. From this value and the standard free energy change of –10.9 kcal mol−1 for the hydrolysis of ATP to AMP and PPi, we calculate that ΔG′o for the hydrolysis of the adenylyl-DNA ligase is –13.2 kcal mol−1. The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pK a of Lys159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pK a 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the α,β-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.