Two adjacent aspartates, Asp 295 and Asp 296, playing major roles in the reaction catalyzed by mouse adenosine deaminase (mADA) were altered using site-directed mutagenesis. These mutants were expressed and purified from an ADA-deficient bacterial strain and characterized. Circular dichroism spectroscopy shows the mutants to have unperturbed secondary structure. Their zinc content compares well to that of wild-type enzyme. Changing Asp 295 to a glutamate decreases the k(cat) but does not alter the K(m) for adenosine, confirming the importance of this residue 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 side chain, resulting in the mutant's inability to turn over the substrate. In contrast, Asp 296 mutants exhibit markedly increased K(m) values, establishing this residue's critical role in substrate binding. The Asp 296 → Ala mutation causes a 70-fold increase in the K(m) for adenosine and retains 0.001% of the wild-type k(cat)/K(m) value, whereas the Asp 296 → Asn mutant has a 10-fold higher K(m) and retains 1% of the 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 single hydrogen bond between a carboxylate oxygen and N7 of the purine ring. These results and others discussed below are in agreement with the postulated role of the adjacent aspartates in the catalytic mechanism for mADA.
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