A Nitroxyl Synthase Catalytic Antibody

Abstract

An antibody raised against acridinium hapten 1 is shown to catalyze the retro Diels−Alder reaction of the anthracene−HNO cycloadduct 2 to release anthracene 4 and nitroxyl (HNO). Nitroxyl is oxidized to nitric oxide (NO) in the presence of superoxide dismutase. Since the enzyme superoxide dismutase is ubiquitous in vivo, this catalytic antibody system may be equivalent to NO-synthase. Antibody catalysis is triggered by recognition of the phenyl rings in hapten 1 at an angle near that of the transition state of the retro Diels−Alder reaction of 2 . Acridinium hapten 1 is in chemical equilibrium with its conjugate Lewis base 9-hydroxyacridane 1 -OH (p K ∼ 8.2). Catalytic antibody 9D9 ( K M ( 2 ) = 100 μM, k cat = 0.07 min - 1, k uncat = 3 × 10 - 4 min - 1 ) is the result of an heterologous immunization and binds both 1 ( K i = 16.6 μM at pH 6.1) and 1 -OH ( K i = 0.9 μM at pH 9.0). The more tightly bound neutral conjugate base 1 -OH probably represents a more accurate mimic of the transition state of the retro Diels−Alder process.