The binding of 1,10‐phenanthroline to specifically active‐site cobalt(II)‐substituted horse‐liver alcohol dehydrogenase

Abstract

We have studied the binding of 1,10‐phenanthroline to specifically active‐site cobalt(II)‐substituted horse‐liver alcohol dehydrogenase [Co(II)‐LADH]. The dissociation constant is a factor of 6500 smaller than in the native enzyme. Spectral evidence is given which shows that 1,10‐phenanthroline does not remove the catalytic Co(II) ion and that binding of 1,10‐phenanthroline renders the catalytic metal ion pentacoordinate. The maximum limiting rate constant for the association of 1,10‐phenanthroline to Co(II)‐LADH is about 60 s⁻¹. This is about a third of the value (169 s⁻¹) determined for native horse‐liver alcohol dehydrogenase, Zn(II)LADH [Frolich et al. (1978) Arch. Biochem. Biophys. 189, 471–480]. For cadmium(II)‐substituted horse‐liver alcohol dehydrogenase, [Cd(II)LADH] the maximum limiting rate constant for association of 1,10‐phenanthroline increased to 590 s⁻¹. These findings demonstrate that the rate‐limiting step is strongly dependent on the chemical nature of the catalytic metal ion and its immediate environment. 1,10‐Phenanthroline is shown to bind to the Co(II)‐LADH · NAD⁺ complex in the open conformation. The maximum limiting rate constant remains unchanged in the presence of NAD⁺. The data have been used to derive a kinetic scheme for the formation of ternary complexes including NAD⁺ that involves a slow intermediary step.