Open AccessActive‐site‐specific zinc‐depleted and reconstituted cobalt(II) human‐liver alcohol dehydrogenase
Author(s) -
SCHNEIDERBERNLÖHR Helga,
FORMICKAKOZŁOWSKA Grażyna,
BÜHLER Rolf,
WARTBURG JeanPierre,
ZEPPEZAUER Michael
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb13995.x
Subject(s) - active site , alcohol dehydrogenase , zinc , chemistry , cofactor , cobalt , isozyme , stereochemistry , ternary complex , enzyme , biochemistry , inorganic chemistry , organic chemistry
The active‐site zinc atom of the β1β1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From β1γ1 and γ1γ1 isozyme the active‐site zinc is extracted much more slowly than from β1β1 isozyme. Only partially active‐site metal‐depleted enzyme species were obtained from these isozymes. The active‐site‐specific reconstituted cobalt(II) derivative of the β1β1 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 β1 and γ1 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.