CHARACTERIZATION OF THE FERROUS ION-BINDING SITES OF APPLE 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE BY SITE-DIRECTED MUTAGENESIS

The putative ferrous ion binding sites (H177, D179, and H234) of apple fruit 1-aminocyclopropane-1-carboxylate oxidase determined by homolog y comparisons with enzymes which required non-haem Fe2+ for activity w ere altered by site-directed mutagenesis. The activities of mutants (H 177F, D179H, D179A, H234F, and H234D) were completely lost. SDS-PAGE a nd western immunoanalysis confirmed that loss of enzyme activity in mu tants was not due to impaired enzyme expression. These results strongl y suggest that H177, D179, and H234 are the Fe2+ binding site. (C) 199 6 Academic Press, Inc.