Hampton A, Patel A D, Chawla R R, Kappler F, Hai T T
J Med Chem. 1982 Apr;25(4):386-92. doi: 10.1021/jm00346a011.
Adenosine 5'-triphosphate (ATP) derivatives of the types N6-R-ATP [R = (CH2)nNHCOCH2I, (CH2)nNHCO-(CH2)mNHCOCH2I, or (CH2)nCON(Me)(CH2)mN(Me)CO(CH2)nNHCOCH2I], N6-Me-N6-R-ATP [R = (CH2)nN-(Me)CO(CH2)mNHCOCH2I], and 8-R-ATP [R = NM(CH2)nNHCOCH2I] with 5--19 spacer atoms between N6 or C-8 and iodine have been evaluated as potential exo-ATP-site-directed reagents for phosphokinases. Substrate and inhibitor properties indicated that the compounds possessed affinity for the ATP sites of the muscle (M), kidney (K), and liver (L) isozymes of rat pyruvate kinase (PK), of E. coli thymidine kinase (TK), and of yeast hexokinase (HK) and rat KH I, II, and III isozymes. Tests for time-dependent loss of enzyme activity (inactivation) were performed under conditions in which a large proportion of each phosphokinase was present as an enzyme-inhibitor complex. No ATP-site-directed inactivations resulted when the M, L, or K isozymes of PK were exposed for 8 h, 22 degrees C, to 5 mM levels of 18 ATP derivatives or 6 analogous ADP derivatives or when yeast HK or rat KH I, II, or III was exposed for 6 h, 22 degrees C, to 5 mM levels of 28 ATP derivatives. Escherichia coli TK was inactivated by 6 of 25 ATP derivatives tested at 10 mM, 6 h, 0 degrees C; inactivation was slowed by MgATP in the case of N6-CH3-N6-R-ATP [R = (CH2)4N(CH3)CO(CH2)5NHCOCH2I]. Only 1% of 298 enzyme-inhibitor combinations exhibited ATP-site-directed inactivation, signifying that few suitably positioned and sufficiently reactive nucleophilic groups were present near the enzymic ATP sites. Studies have now shown that exo-active-site-directed reagents can act as isozyme- or species-selective enzyme inhibitors. The present survey indicates that in many cases such reagents may be difficult of access when data are not available regarding structural or physicochemical features of the target enzyme adjacent to its catalytic site.
已对N6 - R - ATP [R = (CH2)nNHCOCH2I、(CH2)nNHCO - (CH2)mNHCOCH2I或(CH2)nCON(Me)(CH2)mN(Me)CO(CH2)nNHCOCH2I]、N6 - Me - N6 - R - ATP [R = (CH2)nN - (Me)CO(CH2)mNHCOCH2I]和8 - R - ATP [R = NM(CH2)nNHCOCH2I]类型的5'-三磷酸腺苷（ATP）衍生物进行了评估，这些衍生物在N6或C - 8与碘之间有5 - 19个间隔原子，被视为磷酸激酶潜在的外源性ATP位点导向试剂。底物和抑制剂特性表明，这些化合物对大鼠丙酮酸激酶（PK）的肌肉（M）、肾脏（K）和肝脏（L）同工酶、大肠杆菌胸苷激酶（TK）、酵母己糖激酶（HK）以及大鼠KH I、II和III同工酶的ATP位点具有亲和力。在大部分磷酸激酶以酶 - 抑制剂复合物形式存在的条件下，进行了酶活性随时间丧失（失活）的测试。当PK的M、L或K同工酶在22℃下与5 mM浓度的18种ATP衍生物或六种类似的ADP衍生物接触8小时，或者酵母HK或大鼠KH I、II或III在22℃下与5 mM浓度的28种ATP衍生物接触6小时时，未发生ATP位点导向的失活。在0℃下，10 mM浓度下测试的25种ATP衍生物中有6种使大肠杆菌TK失活；对于N6 - CH3 - N6 - R - ATP [R = (CH2)4N(CH3)CO(CH2)5NHCOCH2I]，MgATP减缓了失活。298种酶 - 抑制剂组合中只有1%表现出ATP位点导向的失活，这表明在酶的ATP位点附近存在的合适定位且反应性足够的亲核基团很少。现在的研究表明，外源性活性位点导向试剂可作为同工酶或物种选择性的酶抑制剂。目前的调查表明，在许多情况下，当没有关于靶酶催化位点附近的结构或物理化学特征的数据时，可能难以获得此类试剂。
