Evidence for inner-sphere coordinated alkoxide ion intermediates in the catalytic mechanism of Co(II) substituted liver alcohol dehydrogenase

Abstract

Substitution of certain divalent transition metal ions, i.e. Co(II), Ni(II), Cu(II) and Fe(II), for the active site ion of horse liver alcohol dehydrogenase yields catalytically active enzyme derivatives [1–4]. Since catalysis occurs via inner-sphere coordination of substrate [3,5,6], the ligand-to-metal charge-transfer (LMCT) and d-d electronic transitions of these metal ions are useful spectroscopic probes of the chemical events occurring during catalysis [1, 2, 7]. As will be shown in this report, when reaction is monitored via rapid-scanning, stopped-flow (RSSF) UV-visible spectroscopy, the resulting time-resolved spectra (3000 nm to 700 nm) provide detailed temporal information about transient intermediates formed during catalysis. We report here evidence from RSSF spectroscopy for the existence of ternary enzymeNAD+alkozide ion intermediates both in the Co(II)E-catalyzed reduction of aldehydes and in the Co(II)E-catalyzed oxidation of alcohols.