Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase

Vera Sideraki, Khalid A. Mohamedali, David K. Wilson, Zengyi Chang, Rodney E. Kellems, Florante A. Quiocho, Frederick B. Rudolph

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish (US)
Pages (from-to)7862-7872
Number of pages11
JournalBiochemistry
Volume35
Issue number24
DOIs
StatePublished - Jun 18 1996
Externally publishedYes

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Aspartic Acid
Adenosine
Glutamic Acid
Catalytic Domain
Substrates
Circular Dichroism
Site-Directed Mutagenesis
Zinc
Hydrogen
Spectrum Analysis
Oxygen
Circular dichroism spectroscopy
Mutagenesis
Mutation
Water
Enzymes
Hydrogen bonds
Crystal structure
Mouse Ada protein
purine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sideraki, V., Mohamedali, K. A., Wilson, D. K., Chang, Z., Kellems, R. E., Quiocho, F. A., & Rudolph, F. B. (1996). Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase. Biochemistry, 35(24), 7862-7872. https://doi.org/10.1021/bi952920d

Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase. / Sideraki, Vera; Mohamedali, Khalid A.; Wilson, David K.; Chang, Zengyi; Kellems, Rodney E.; Quiocho, Florante A.; Rudolph, Frederick B.

In: Biochemistry, Vol. 35, No. 24, 18.06.1996, p. 7862-7872.

Research output: Contribution to journalArticle

Sideraki, V, Mohamedali, KA, Wilson, DK, Chang, Z, Kellems, RE, Quiocho, FA & Rudolph, FB 1996, 'Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase', Biochemistry, vol. 35, no. 24, pp. 7862-7872. https://doi.org/10.1021/bi952920d
Sideraki V, Mohamedali KA, Wilson DK, Chang Z, Kellems RE, Quiocho FA et al. Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase. Biochemistry. 1996 Jun 18;35(24):7862-7872. https://doi.org/10.1021/bi952920d
Sideraki, Vera ; Mohamedali, Khalid A. ; Wilson, David K. ; Chang, Zengyi ; Kellems, Rodney E. ; Quiocho, Florante A. ; Rudolph, Frederick B. / Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase. In: Biochemistry. 1996 ; Vol. 35, No. 24. pp. 7862-7872.
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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 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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AU - Quiocho, Florante A.

AU - Rudolph, Frederick B.

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