Reactivation of apo horse liver alcohol dehydrogenase with the monovalent metal ion Ag(I)

1982 ◽  
Vol 2 (7) ◽  
pp. 509-514 ◽  
Author(s):  
Lars Skjeldal ◽  
Knut H. Dahl ◽  
John S. McKinley-McKee
Keyword(s):  
Alcohol Dehydrogenase ◽  
Metal Binding ◽  
Metal Ion ◽  
Specific Activity ◽  
Native Enzyme ◽  
Catalytic Center ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
Affinity Labelling ◽  
Catalytic Metal

Each subunit of the liver alcohol dehydrogenase dimer contains one catalytic and one structural Zn(II) atom. Enzyme with the catalytic metal atoms selectively removed is inactive but can be partly reactivated in the presence of Ag(I) ions. Reactivation results from Ag(1) ions entering the empty metal-binding site in the catalytic center. The specific activity of this silver enzyme reached 24% of the native enzyme. Atomic absorption analysis gave equal amounts of Ag(I) and Zn(II), corresponding to one mole of each metal per monomer. Metal-directed affinity labelling using bromo-imidazolyl propionate showed that the properties of the silver-reactivated enzyme were distinct from those of the native enzyme.

scholarly journals Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme

1983 ◽  
Vol 211 (2) ◽  
pp. 391-396 ◽  
Author(s):  
K H Dahl ◽  
H Eklund ◽  
J S McKinley-McKee
Keyword(s):  
Alcohol Dehydrogenase ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Inactivation Kinetics ◽  
Horse Liver Alcohol Dehydrogenase ◽  
Three Dimensional Structure ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
Affinity Labelling

Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S-2-chloro-3-(imidazol-5-yl)propionate at pH8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity.

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