Probing the active site of adenosine deaminase by a pH responsive fluorescent competitive inhibitor

Valeria R. Caiolfa, David Gill, Abraham H. Parola

Research output: Contribution to journalArticle

Abstract

The adenine analog erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA, a tight reversible inhibitor (K(I) = 1.6 x 10-9 M) of adenosine deaminase (EC 3.5.4.4) (ADase), was modified into the fluorescent etheno derivative ε-EHNA. The latter is a competitive inhibitor of adenosine deaminase [K(I) = (2.80 ± 0.01)10-6 M], having the fluorescent properties of ε-adenines. Affinity to the active site, monitored by both steady-state and dynamic fluorescence polarization, was confirmed by competition experiments with 2'-deoxycoformycin, the substrate adenosine and EHNA. The ε-adenine moiety of ε-EHNA librates at the shallow active site of ADase. The low absorptivity of ε-EHNA required the measurement of fluorescence excitation spectra. Computer subtraction of fluorescence excitation spectrum of ADase from that of its equimolar complex with ε-EHNA revealed the corrected excitation spectrum of ε-EHNA at the active site of the enzyme. This spectrum mimics that of ε-EHNA at pH 5.5 in buffer solution, implying its protonation at the active site of the enzyme. These results are in agreement with the presence of acidic amino acids that are essential to the catalytic mechanism.

Original languageEnglish
Pages (from-to)41-56
Number of pages16
JournalBiophysical Chemistry
Volume70
Issue number1
DOIs
Publication statusPublished - Jan 1 1998

Keywords

  • Adenosine deaminase
  • Etheno analogues
  • Etheno-EHNA
  • Fluorescent competitive inhibitor
  • pH sensitive inhibitor of ADase
  • Protonation equilibrium

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Physical and Theoretical Chemistry

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