Towards the Mechanisms of AlkA-Catalyzed and Acid-Catalyzed 2’-Deoxyinosine Hydrolysis: Investigations Into Leaving-Group Activation and Transition State Structure

Abstract

Escherichia coli AlkA (3-methyladenine DNA glycosylase II) is a DNA repair enzyme that can excise both damaged and undamaged nucleobases. To determine whether AlkA’s excision of neutral purines is facilitated by general- acid catalysis (i.e., protonation of the nucleobase), pH vs. kcat and pH vs. kcat/KM profiles for hypoxanthine excision were generated. Each profile revealed acid catalysis and a single pKa of 5.7 ± 0.1 and 5.1 ± 0.2, respectively. Mutants of ionizable and hydrogen-bonding active site residues – Y273F, W272F, Y222F, R244M, and R22M – demonstrated at most a 4-fold reduction in hypoxanthine excision, so these residues were not involved in purine protonation. A dependence on buffer concentration was observed in the mutants at pH 7 and in wild-type AlkA at pH 7 and 8 but not at pH < 7. Solvent deuterium kinetic isotope effects (KIEs) at pH 6 and 7 showed that proton transfer was rate-limiting at pH 7 when [DNA] > KM (KM = 1.0 μM). AlkA could not bind adenine, hypoxanthine, and nicotinamide along with a transition state-mimicking pyrrolidine DNA. Therefore, AlkA does not interact specifically with the nucleobase to protonate it. To determine the structure of AlkA’s transition state, methods for primary and secondary KIE measurement were developed, but none of the methods produced precise KIE values (95% confidence interval < 0.005). To determine the structure of the transition state of the cognate non-enzymatic reaction, the acid-catalyzed hydrolysis of 2’-deoxyinosine 5’-monophosphate, the 1’-3H, 4’-3H, 5’-3H2, 1’-14C, and 7-15N KIEs were measured and were 1.25 ± 0.02, 0.94 ± 0.01, 0.99 ± 0.02, 0.997 ± 0.004, and 0.99 ± 0.05, respectively. Despite being consistent with an oxacarbenium ion-like transition state, these values would not be precise enough to distinguish between potential transition state models.