THE PROTONATED FORM OF 1-N6-ETHENO-[ERYTHRO-9-(2-HYDROXY-3-NONYL)] ADENINE IS IDENTIFIED AT THE ACTIVE-SITE OF ADENOSINE-DEAMINASE

A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), 1-N6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (ε{lunate}-EHNA), is protonated at the active site of the enzyme. In ε{lunate}-EHNA [K1 = (4.06 ± 1.00) 10-6 M] part of the competive inhibition of EHNA is combined with spectroscopic properties of etheno-adenines. Computer subtraction of the fluorescence excitation spectrum of ADA from that of its equimolar complex with ε{lunate}-EHNA yielded the corrected excitation spectrum of ε{lunate}-EHNA at the active site of the enzyme. This spectrum mimics that of ε{lunate}-EHNA at pH 5.5 in buffer solution and is suggested to indicate a shift in protonation equilibrium at the active site. © 1990.