Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites

Abstract

1. The zinc‐deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13‐ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1‐anilinonaphthalene 8‐sulphonic acid is bound with comparable affinity. 2. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc‐deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. 3. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 4. 2,2′‐Bipyridine, which chelates the active‐site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc‐depleted enzyme. 5. The stopped‐flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc‐depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His‐67, bound to the zinc ion, or the zinc‐liganding water molecule in the case of the native enzyme (pK close to 9), or the free His‐67 residue in the case of the zinc‐deficient enzyme (pK about 8).

Differential Effects of the Zn−His−Bkb vs Zn−His−[Asp/Glu] Triad on Zn-Core Stability and Reactivity

“…Apart from playing a structural role, Zn 2+ can also play a regulatory role in electron transfer, substrate oxidation−reduction, and transport processes. Furthermore, Zn 2+ can play a catalytic role in (i) facilitating the ionization of a first-shell water molecule to a nucleophilic hydroxide ion in proteins such as carbonic anhydrase , and metallo-β-lactamase or (ii) stabilizing the negatively charged intermediate of enzymes such as carboxypeptidase A , and alcohol dehydrogenase. , …”

“…Furthermore, Zn 2+ can play a catalytic role in (i) facilitating the ionization of a first-shell water molecule to a nucleophilic hydroxide ion in proteins 20 such as carbonic anhydrase 21,22 and metallo-β-lactamase 23 or (ii) stabilizing the negatively charged intermediate of enzymes such as carboxypeptidase A 24,25 and alcohol dehydrogenase. 26,27 Interestingly, a survey of Zn-proteins in the Protein Data Bank 28,29 (PDB) has shown that catalytic and structural zinc † Academia Sinica. ‡ National Tsing Hua University.…”