Additional insights into the structure and function of adenosine deaminase
Martinez, Jeannette Huczko
Rudolph, Frederick B.
Doctor of Philosophy
Adenosine deaminase is a key enzyme in purine catabolism. The elucidation of the crystal structure and recent analysis of site-directed mutants have provided details on the structure and function of the enzyme. The studies described in this thesis provide new insights into ADA. The ADA mechanism is well defined except for the identification of the proton acceptor. Aspartate 295 is important to metal coordination (Wilson, 1991). This residue may be the catalytic base because of its position in the original crystal structure. Mutation of this residue to asparagine resulted in a 100,000 fold reduction in activity but retention of binding to substrates and inhibitors. UV difference spectra confirm that the tetrahedral intermediate is not formed during catalysis. These results suggest that Asp295 functions as the proton acceptor and in metal coordination. The crystal structure of mouse ADA provides a static picture of the enzyme in a single conformation. The sequestered nature of the active site indicates that the motion of one or more side chains is necessary for the proper functioning of the enzyme. Several mutants were created involving a flexible loop at residues 109-124. Complete excision, mutation to a hinge, or the replacement of the loop with the smaller loop found in E. coli ADA resulted in decreases in activity. Binding was not affected by any of these mutations. The 109-124 loop most likely acts in sequestering the active site from solvent during catalysis. An alanine to valine point mutation has been located in codon 329 of patients with SCID. Site directed mutagenesis was used to generate this mutation. Protein containing this change was generated and was found to be misfolded and catalytically inactive. The loop mutations, A329V, and D295N affect the rate of catalysis of ADA for three different reasons. The loop serves to shield the active site from solvent. Asp295 is the catalytic base. Ala329 is important to protein folding. These studies show how slight alterations in primary structure can affect the overall function of an enzyme.