Hixson C S, Krebs E G
J Biol Chem. 1979 Aug 25;254(16):7509-14.
Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.
5'-对氟磺酰苯甲酰腺苷与牛心肌环磷酸腺苷依赖性蛋白激酶的催化亚基一起温育，导致形成一种失活的酶，每摩尔亚基大约被一摩尔试剂不可逆地修饰。失活反应遵循假一级动力学。在不同试剂浓度下的失活速率呈现出饱和动力学，这意味着该试剂在失活之前可逆地与酶结合。向反应混合物中添加MgATP、MgADP或MgAMP-PNP可完全保护酶不被5'-对氟磺酰苯甲酰腺苷失活。已证明该试剂是MgATP的竞争性抑制剂，其Ki为0.235 mM。无金属核苷酸对反应速率没有影响，而单独的金属离子可将失活速率加快至7倍。在温育混合物中加入酪蛋白或合成肽底物不影响反应动力学。5'-对氟磺酰苯甲酰腺苷与激酶亚基的反应表现出MgATP结合位点亲和标记的所有特征。
