The goals of the studies described in this proposal are to understand the relationship between molecular structure and functional properties of adenosine deaminase (ADA) and related proteins. ADA is present in all mammalian cells and tissues but the level of the enzyme varies by more than 10,000 fold in a tissue specific manner. ADA has a central role in development of the lymphoid system as genetic absence of the enzyme leads to severe combined immunodeficiency disease and its levels are related to other immunological and neoplastic disease states. ADA function is critical in control of the effects of adenosine in a variety of systems including roles in neurological function, catabolism of nutrients and the vascular system. A number of clinically important purine analogues are used as inhibitors of ADA including 2-deoxycoformycin (pentostatin) and several neurologically active compounds. This enzyme represents an ideal system for detailed analysis as it is available in large amounts in a stable form isolated from cloned genes, can easily be mutated, catalyzes a simple, important reaction and is a relatively small monomeric protein with a zinc cofactor. The crystal structure of the enzyme with a transition state analogue has been determined and a new catalytic mechanism involving the newly discovered zinc atom at the active site has been proposed. The requirement for zinc may be a part of the effect of dietary zinc deficiency on suppression of the immune system. Additional enzymes with similar catalytic function are available to evaluate the general nature of this work. Specific proposed studies include: 1. Identification and functional analysis of specific amino acid residues involved in the catalytic process for ADA. 2. Use of metal substituted ADA to study substrate and inhibitor interactions with the enzyme. 3. Identification and functional analysis of specific amino acid residues involved in the catalytic mechanism of AMP deaminase by comparison of conserved sequences between ADA and AMP deaminase. The results from these studies will provide a clear basis for understanding how ADA functions as a catalyst along with other enzymes with similar mechanisms such as AMP deaminase.
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