Biochemical effects of adenosine (Ado) and 2'-deoxyadenosine (dAdo) block lymphoid development and cause severe immune dysfunction in genetic deficiency of Ado deaminase (ADA). One target of these nucleosides is the essential metabolic enzyme S-adenosylhomocysteine hydrolase (AdoHcyase), which we identified as a high affinity Ado and cyclic AMP binding protein. Ado inhibits AdoHcy hydrolysis in ADA deficient cells. We discovered that dAdo irreversibly inactivates AdoHcyase by causing reduction of enzyme associated NAD. In ADA deficiency these effects of Ado and dAdo result in secondary deficency of AdoHcyase and accumulation of AdoHcy, which is an inhibitor of S-adenosylmethionine (AdoMe+) dependent transmethylation reactions. We recently showed that the genetic loci for AdoHcyase and ADA are linked, suggesting an evolutionary relationship between these enzymes. We propose to examine the possible relatedness of AdoHycase to ADA or to other proteins that bind NAD, Ado and cAMP; to provide information, which is currenty unavailable, regarding the primary sequence and structure of AdoHcyase and the nature of its catalytic and ligand binding sites; to investigate the influence of ADA deficiency on the turnover of AdoHcyase; and to continue studies now in progess of the metabolic consequences to lymphoid cells of the inhibition of AdoHcy hydrolysis. Specifically, we will attempt to isolate and sequence the cDNA for human AdoHcyase mRNA, using monoclonal antibodies to AdoHcyase and AdoHcyase specific oligonucleotides as probes for screening human liver cDNA libraries. We will evaluate the possibility that Adohcyase may be composed of two kinds of subunits. Essential amino acid residues involved in ligand binding and in catalysis will be located within the sequence of AdoHcyase. We will attempt to crystallize AdoHcyase in the presence and absence of ligands so that its structure may be established definitively. Antibodies and cDNA probes will be used to determine whether inactivation of AdoHcyase by dAdo in ADA deficiency alters the rate of AdoHCyase degradation or AdoHcyase turnover. We will characterize lymphoblast mutants that we have selected for resistance to Ado analogues that specifically inhibit Adohcyase, with the aim of better defining the effect of AdoHcy accumulation on the synthesis and turnover of AdoMet.
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