S-Adenosylhomocysteine hydrolase is the only enzyme in eukaryotic cells for the removal of adenosylhomocysteine, the end-product of biological trans- methylation reactions. For this reason, the enzyme is critical for the regulation of adenosylmethionine-dependent methylations. We have used several approaches to investigate structure/function relationships of AdoHcy hydrolase. We have cloned the cDNA for the enzymes from both rat liver and D. discoideum, and the enzymes have been expressed in E. coli. The amino acid sequence is highly conserved between species suggesting that much of the sequence is required for enzyme function. The putative NAD binding site has been identified by homology to several dehydrogenases and by site-directed mutagenesis of specific amino acids within this region. Investigation of the inactivation of the rat liver enzyme by the site- specific reagent, fluorosulfonylbenzol-adenosine, yielded data that support the role of a specific cysteine (cysteine 78) in enzyme function. During inactivation of the enzyme a disulfide between cysteine 78 and cysteine 112 is formed that can be reduced with a thiol to reactivate the enzyme. Formation of the disulfide indicates that cysteines 78 and 112 are near each other in the three dimensional structure of the protein. The reactivity of fluorosulfonylbenzoyladenosine with site-directed mutants of cysteines 78 and 112 have provided specific details of the chemical reactions that occur in the formation of the disulfide.
