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

The adenine analog erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA, a tight reversible inhibitor (KI = 1.6 x 10(-9) M) of adenosine deaminase (EC 3.5.4.4) (ADase), was modified into the fluorescent etheno derivative epsilon-EHNA. The latter is a competitive inhibitor of adenosine deaminase [KI = (2.80 +/- 0.01)10(-6) M], having the fluorescent properties of epsilon-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 epsilon-adenine moiety of epsilon-EHNA librates at the shallow active site of ADase. The low absorptivity of epsilon-EHNA required the measurement of fluorescence excitation spectra. Computer subtraction of fluorescence excitation spectrum of ADase from that of its equimolar complex with epsilon-EHNA revealed the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-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.