Sarfati R S, Kansal V K, Munier H, Glaser P, Gilles A M, Labruyère E, Mock M, Danchin A, Bârzu O
Unités de Chimie Organique, Institut Pasteur, Paris, France.
J Biol Chem. 1990 Nov 5;265(31):18902-6.
3'-Anthraniloyl-2'-deoxyadenosine 5'-triphosphate (Ant-dATP), a fluorescent analogue of ATP, was tested as a probe for the nucleotide-binding site of calmodulin (CaM)-activated adenylate cyclases from Bordetella pertussis (BPCYA47) and Bacillus anthracis (BACYA62). Ant-dATP competitively inhibited both bacterial enzymes expressed in Escherichia coli (ki approximately 10 microM). Binding of the analogue to adenylate cyclase was monitored by equilibrium dialysis and by an increase in its fluorescence emission at 420 nm upon excitation at 330 nm. Whereas the fluorescence of Ant-dATP was little influenced by divalent cations, CaM, or adenylate cyclase alone, the Ca2+.CaM.cyclase complex increased up to 4 times the quantum yield of Ant-dATP. Binding of the analogue to the catalytic site of BPCYA47 and BACYA62 was specific as shown by its displacement with ATP or 3'-dATP. Our results substantiate the role of CaM in favoring substrate binding to CaM-activated enzymes.
3'-邻氨基苯甲酰基-2'-脱氧腺苷5'-三磷酸（Ant-dATP），一种ATP的荧光类似物，被用作检测来自百日咳博德特氏菌（BPCYA47）和炭疽芽孢杆菌（BACYA62）的钙调蛋白（CaM）激活的腺苷酸环化酶核苷酸结合位点的探针。Ant-dATP竞争性抑制在大肠杆菌中表达的两种细菌酶（抑制常数约为10微摩尔）。通过平衡透析以及在330纳米激发时其在420纳米处荧光发射的增加来监测该类似物与腺苷酸环化酶的结合。虽然Ant-dATP的荧光单独受二价阳离子、CaM或腺苷酸环化酶的影响很小，但Ca2⁺·CaM·环化酶复合物使Ant-dATP的量子产率提高了多达4倍。如ATP或3'-dATP对其的置换所示，该类似物与BPCYA47和BACYA62催化位点的结合具有特异性。我们的结果证实了CaM在促进底物与CaM激活的酶结合中的作用。
