Active‐site‐specific zinc‐depleted and reconstituted cobalt(II) human‐liver alcohol dehydrogenase

Publisher Website
Google Scholar
Abstract
The active-site zinc atom of the .beta.1.beta.1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From .beta.1.gamma.1 and .gamma.1.gamma.1 isozyme the active-site zinc is extracted much more slowly than from .beta.1.beta.1 isozyme. Only partially active-site metal-depleted enzyme species were obtained from these isozymes. The active-site-specific reconstituted cobalt(II) derivative of the .beta.1.beta.1 isozyme shows spectroscopic properties comparable to those of the active-site-specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme-induced conformational change of the protein leads to a red shift of the d-d band from 648 nm to 673 nm. The chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between .beta.1 and .gamma.1 subunits (threonine-48 instead of serine-48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal-bound chromophoric substrate.