SITE-DIRECTED MUTAGENESIS OF ACTIVE-SITE GLUTAMATE-217 IN MOUSE ADENOSINE-DEAMINASE

Mouse adenosine deaminase (ADA) contains an active site glutamate resi due at position-217 that is highly conserved in other adenosine and AM P deaminases. Previous research has suggested that proton donation to N-1 of the adenosine ring occurs prior to catalysis and supports the m echanism as proceeding via formation of a tetrahedral intermediate at C-6 of adenosine. The proposed catalytic mechanism of ADA based on the recent elucidations of the crystal structure of this enzyme with tran sition- and ground-state analogs hypothesized that GlU(217) was involv ed in this proton donation step [Wilson, D. K., Rudolph, F. B., & Quio cho, F. A. (1991) Science 252, 1278-1284; Wilson, D. K., & Quiocho, F. A. (1993) Biochemistry 32, 1689-1693]. Site-directed mutagenesis of t he equivalent glutamate in human ADA resulted in a dramatic loss of en zyme activity [Bhaumik, D., Medin, J., Gathy, K., gr Coleman, M. (1993 ) J. Biol. Chem. 268, 5464-5470]. To further study the importance of t his residue, site-directed mutagenesis was used to create mouse ADA mu tants. Glu(217) was mutated to Asp, Gly, Gin, and Ser, and all mutants were successfully expressed and purified. Circular dichroism and zinc analysis showed no significant changes in secondary structure or zinc content, respectively, compared to the native protein. The mutants sh owed only a slight variation in K-m but dramatically reduced k(cat) le ss than 0.2% of wildtype activity. UV difference and C-13 NMR spectra conclusively demonstrated the failure of any of these mutants to hydra te purine riboside, a reaction carried out by the wild-type enzyme tha t results in formation of an enzyme-inhibitor complex. Surprisingly, K -i values for binding of the inhibitor to the mutants and to wild-type protein are similar, irrespective of whether the inhibitor is hydrate d upon binding. These data confirm the importance of Glu(217) in catal ysis as suggested by the crystal structure of mouse ADA.