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-III alcohol dehydrogenase have been exchanged by site-directed mutagenesis in order to study the importance of the metal in the mammalian enzymes. The cysteine residues were replaced with Ala and Ser, residues that are not able to ligand zinc. All mutations resulted in inactive, unstable enzymes, in contrast to the non-mutated human alcohol dehydrogenases that are easily isolated. Northern-blot analysis revealed the presence of the expected mRNAs from expression plasmids constructed with the different mutated and non-mutated alcohol dehydrogenases, and Western-blot analysis gave faint signals for the mutated recombinant proteins from crude extracts. This verifies that the plasmid constructs are correct, but that the translated, mutated proteins lacking the zinc-stabilized local fold, are subject to rapid degradation. Hence, the results directly illustrate the importance of the structural zinc atom in mammalian alcohol dehydrogenase and confirm it as a component with 'structural' properties. The results are compatible with those from sensitivities to proteases and from the structures of other proteins within the super-family, indicating that the structural role of the zinc atom may involve conservation of interfaces regulating the enzyme quaternary structure.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
J
J
Jeloková
C
C
Karlsson
M
M
Estonius
H
H
Jörnvall
J O
JO
Höög
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015032:Zinc / Q000737:chemistry
D017931:DNA Primers; D008024:Ligands; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000426:Alcohol Dehydrogenase; D015032:Zinc; D003545:Cysteine