Kinetic and Mechanistic Studies of Human Oligoadenylate Synthetase 1.

Abstract

Oligoadenylate synthetase 1 (OAS1) catalyzes the dsRNA-dependent polymerization of ATP to form oligoadenylate, a second messenger of the innate immunity system. This paper reports kinetic and mechanistic studies of OAS1-catalyzed dimerization of ATP to form 2'-5'-diadenylate and pyrophosphate (PPi), the first step in ATP polymerization. Major findings include the following: (1) Reaction progress curves for the production of PPi are biphasic, characterized by a presteady-state lag followed by the linear, steady-state production of PPi. (2) The dependence of steady-state velocity on ATP concentration is sigmoidal and can be described by a rate law derived for a mechanism involving enzyme-catalyzed substrate dimerization. (3) Steady-state velocities were determined as a function of ATP concentration at fixed concentrations of poly(I:C), a synthetic dsRNA activator of OAS1. The data suggest a random mechanism in which either ATP or poly(I:C) can add first to the enzyme. (4) The dependence of klag on poly(I:C) and ATP concentration requires expansion of this mechanism to include slow conformational isomerization of various poly(I:C)- and ATP-bound complexes of inactive OAS1 to form complexes comprising an active enzyme, to ultimately form the reactive Michaelis complex of active OAS1, poly(I:C), and two molecules of ATP. Finally, within this complex, the two molecules of ATP dimerize to form 2'-5'-diadenylate and pyrophosphate. (5) The pH dependence and solvent deuterium isotope effect for kcat suggests that proton transfer occurs in the rate-limiting transition state, which likely involves proton abstraction from the 2'-hydroxyl of the adenylate acceptor ATP as the oxygen of this hydroxyl attacks the a-phosphate of the adenylate donor ATP in an SN2 fashion.