Active-site-specific zinc-depleted and reconstituted cobalt(II) human-liver alcohol dehydrogenase. Preparation, characterization and complexation with NADH and trans-4-(N,N-dimethylamino)-cinnamaldehyde.
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.
Schneider-Bernlöhr
H
H
Fachrichtung 14.4-Biochemie, Universität des Saarlandes, Saarbrücken, Federal Republic of Germany.
Formicka-Kozłowska
G
G
Bühler
R
R
von Wartburg
J P
JP
Zeppezauer
M
M
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification*; D000818:Animals; D001665:Binding Sites; D002934:Cinnamates / Q000032:analysis*; D002942:Circular Dichroism; D003035:Cobalt / Q000032:analysis*; D006736:Horses; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000032:analysis*; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D015032:Zinc / Q000032:analysis*
D002934:Cinnamates; D007527:Isoenzymes; D003035:Cobalt; D009249:NADP; C018523:4-dimethylaminocinnamaldehyde; D000426:Alcohol Dehydrogenase; D015032:Zinc