| Abstract
| - Mouse adenosine deaminase (ADA) contains an active site glutamateresidue at position-217that is highly conserved in other adenosine and AMP deaminases.Previous research has suggested thatproton donation to N-1 of the adenosine ring occurs prior to catalysisand supports the mechanism asproceeding via formation of a tetrahedral intermediate at C-6 ofadenosine. The proposed catalyticmechanism of ADA based on the recent elucidations of the crystalstructure of this enzyme with transition-and ground-state analogs hypothesized that Glu217 wasinvolved in this proton donation step [Wilson, D.K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252,1278−1284; Wilson, D. K., & Quiocho, F. A.(1993) Biochemistry 32, 1689−1693]. Site-directedmutagenesis of the equivalent glutamate in humanADA resulted in a dramatic loss of enzyme activity [Bhaumik, D.,Medin, J., Gathy, K., & Coleman, M.(1993) J. Biol. Chem. 268, 5464−5470]. Tofurther study the importance of this residue,site-directedmutagenesis was used to create mouse ADA mutants.Glu217 was mutated to Asp, Gly, Gln, and Ser,andall mutants were successfully expressed and purified. Circulardichroism and zinc analysis showed nosignificant changes in secondary structure or zinc content,respectively, compared to the native protein.The mutants showed only a slight variation inKm but dramatically reducedkcat, less than 0.2% of wild-type activity. UV difference and 13C NMR spectraconclusively demonstrated the failure of any of thesemutants to hydrate purine riboside, a reaction carried out by thewild-type enzyme that results in formationof an enzyme−inhibitor complex. Surprisingly,Ki values for binding of the inhibitor to themutants andto wild-type protein are similar, irrespective of whether the inhibitoris hydrated upon binding. Thesedata confirm the importance of Glu217 in catalysis assuggested by the crystal structure of mouse ADA.
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