PROBING THE FUNCTIONAL-ROLE OF 2 CONSERVED ACTIVE-SITE ASPARTATES IN MOUSE ADENOSINE-DEAMINASE

Two adjacent aspartates, Asp 295 and Asp 296, playing major roles in t he reaction catalyzed by mouse adenosine deaminase (mADA) were altered using site-directed mutagenesis. These mutants were expressed and pur ified from an ADA-deficient bacterial strain and characterized. Circul ar dichroism spectroscopy shows the mutants to have unperturbed second ary structure. Their zinc content compares well to that of wild-type e nzyme. Changing Asp 295 to a glutamate decreases the k(cat) but does n ot alter the K-m for adenosine, confirming the importance of this resi due in the catalytic process and its minimal role in substrate binding . The crystal structure of the D295E mutant reveals a displacement of the catalytic water from the active site due to the longer glutamate s ide chain, resulting in the mutant's inability to turn over the substr ate. In contrast, Asp 296 mutants exhibit markedly increased K-m value s, establishing this residue's critical role in substrate binding. The Asp 296 --> Ala mutation causes a 70-fold increase in the K-m for ade nosine and retains 0.001% of the wild-type k(cat)/K-m value, whereas t he Asp 296 --> Asn mutant has a 10-fold higher K-m and retains 1% of t he wild-type k(cat)/K-m value. The structure of the D296A mutant shows that the impaired binding of substrate is caused by the loss of a sin gle hydrogen bond between a carboxylate oxygen and N7 of the purine ri ng. These results and others discussed below are in agreement with the postulated role of the adjacent aspartates in the catalytic mechanism for mADA.