The protonated form of 1‐N 6‐etheno‐[erythro‐9‐(2‐hydroxy‐3‐nonyl)] adenine is identified at the active site of adenosine deaminase

[摘要]

A novel fluorescent competitive inhibitor of adenosine deaminase (EC 3.5.4.4) (ADA), 1-N ⁶-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine (ϵ-EHNA), is protonated at the active site of the enzyme. In ϵ-EHNA [K ₁ = (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-EHNA yielded 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 and is suggested to indicate a shift in protonation equilibrium at the active site.

[学科分类] 生物化学/生物物理

[关键词] Adenosine deaminase;1-N 6-Etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine;Etheno-adenine;Fluorescent inhibitor;pH indicator;Fluorescence polarization;ADA;adenosine deaminase;ϵ-EHNA;1-N 6-etheno-[erythro-9-(2-hydroxy-3-nonyl)] adenine;EHNA;erythro-9-(2-hydroxy-3-nonyl)] adenine;2'-deoxycoformycin (Pentostatin);[3-(2'-deoxy-β-D-erythro-pentofuranosyl)-3;6;7;8-tetrahydroimidazol[4;5-d][1;3]diazepin-8-(R)-ol]. The abbreviation ‘ϵ’ was introduced by Secrist et al. [14] and stands for etheno