The role of divalent cations in structure and function of murine adenosine deaminase

Bruce F. Cooper, Vera Sideraki, David K. Wilson, David Y. Dominguez, Sandra W. Clark, Florante A. Quiocho, Frederick B. Rudolph

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6-methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe2+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (K(i) ≤ 1000 μM) was noted for Ca2+, Fe2+, and Fe3+.

Original languageEnglish (US)
Pages (from-to)1031-1037
Number of pages7
JournalProtein Science
Volume6
Issue number5
StatePublished - 1997
Externally publishedYes

Fingerprint

Apoenzymes
Adenosine Deaminase
Divalent Cations
Holoenzymes
Restoration
Zinc
Substrates
Fluorescence
Metals
Pentostatin
Dialysis
Chelating Agents
Cobalt
Tryptophan
Adenosine
Metal ions
Cations
Quenching
Buffers
Binding Sites

Keywords

  • adenosine deaminase
  • apoenzyme
  • divalent cation
  • kinetic studies
  • metal inhibition
  • metal substitution
  • metalloenzyme
  • structural role of metal

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cooper, B. F., Sideraki, V., Wilson, D. K., Dominguez, D. Y., Clark, S. W., Quiocho, F. A., & Rudolph, F. B. (1997). The role of divalent cations in structure and function of murine adenosine deaminase. Protein Science, 6(5), 1031-1037.

The role of divalent cations in structure and function of murine adenosine deaminase. / Cooper, Bruce F.; Sideraki, Vera; Wilson, David K.; Dominguez, David Y.; Clark, Sandra W.; Quiocho, Florante A.; Rudolph, Frederick B.

In: Protein Science, Vol. 6, No. 5, 1997, p. 1031-1037.

Research output: Contribution to journalArticle

Cooper, BF, Sideraki, V, Wilson, DK, Dominguez, DY, Clark, SW, Quiocho, FA & Rudolph, FB 1997, 'The role of divalent cations in structure and function of murine adenosine deaminase', Protein Science, vol. 6, no. 5, pp. 1031-1037.
Cooper BF, Sideraki V, Wilson DK, Dominguez DY, Clark SW, Quiocho FA et al. The role of divalent cations in structure and function of murine adenosine deaminase. Protein Science. 1997;6(5):1031-1037.
Cooper, Bruce F. ; Sideraki, Vera ; Wilson, David K. ; Dominguez, David Y. ; Clark, Sandra W. ; Quiocho, Florante A. ; Rudolph, Frederick B. / The role of divalent cations in structure and function of murine adenosine deaminase. In: Protein Science. 1997 ; Vol. 6, No. 5. pp. 1031-1037.
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