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Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase
V Sideraki1, K A Mohamedali, D K Wilson
1Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
Abstract:
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 kcat but does not alter the Km 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 Km values, establishing this residue's critical role in substrate binding. The Asp 296->Ala mutation causes a 70-fold increase in the Km for adenosine and retains 0.001% of the wild-type kcat/Km value, whereas the ASP 296->Asn mutant has a 10-fold higher Km and retains 1% of the wild-type kcat/Km 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.
Insights
Mouse adenosine deaminase (mADA) function was investigated by altering key aspartate residues. Asp 295 is crucial for catalysis, while Asp 296 is vital for substrate binding, impacting enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Adenosine deaminase (ADA) is a critical enzyme in purine metabolism.
- Specific residues, Asp 295 and Asp 296, are hypothesized to be key in mouse ADA (mADA) catalysis.
- Understanding these residues' roles is essential for elucidating mADA's reaction mechanism.
Purpose of the Study:
- To investigate the functional roles of Asp 295 and Asp 296 in mouse adenosine deaminase (mADA).
- To determine the impact of mutations at these positions on enzyme kinetics and structure.
- To clarify the catalytic mechanism of mADA through site-directed mutagenesis.
Main Methods:
- Site-directed mutagenesis was used to create Asp 295 and Asp 296 mutants of mADA.
- Mutant enzymes were expressed, purified, and characterized using circular dichroism spectroscopy and zinc content analysis.
- Enzyme kinetics (kcat, Km) were measured, and crystal structures of key mutants were determined.
Main Results:
- Mutating Asp 295 to glutamate (D295E) impaired catalysis (decreased kcat) but not substrate binding (Km unchanged), with structural data showing displaced catalytic water.
- Mutations at Asp 296 (D296A, D296N) significantly increased Km, indicating a critical role in substrate binding.
- The D296A structure revealed loss of a hydrogen bond essential for substrate binding, explaining the reduced affinity.
Conclusions:
- Asp 295 is primarily involved in the catalytic step of the mADA reaction.
- Asp 296 plays a crucial role in substrate binding through hydrogen bonding interactions.
- These findings support the postulated mechanism involving adjacent aspartates in mADA's catalytic process.