FEATURES OF STRUCTURAL ZINC IN MAMMALIAN ALCOHOL-DEHYDROGENASE - SITE-DIRECTED MUTAGENESIS OF THE ZINC LIGANDS

All four cysteine ligands to the structural zinc atom of human class-I and class-m alcohol dehydrogenase have been exchanged by site-directe d mutagenesis in order to study the importance of the metal in the mam malian enzymes. The cysteine residues were replaced with Ala and Ser, residues that are not able to ligand zinc. All mutations resulted in i nactive, unstable enzymes, in contrast to the non-mutated human alcoho l dehydrogenases that are easily isolated. Northern-blot analysis reve aled the presence of the expected mRNAs from expression plasmids const ructed with the different mutated and non-mutated alcohol dehydrogenas es, and Western-blot analysis gave faint signals for the mutated recom binant proteins from crude extracts. This verifies that the plasmid co nstructs are correct, but that the translated, mutated proteins lackin g the zinc-stabilized local fold, are subject to rapid degradation. He nce, the results directly illustrate the importance of the structural zinc atom in mammalian alcohol dehydrogenase and confirm it as a compo nent with 'structural' properties. The results are compatible with tho se from sensitivities to proteases and from the structures of other pr oteins within the super-family, indicating that the structural role of the zinc atom may involve conservation of interfaces regulating the e nzyme quaternary structure.