P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites
P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites
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DOI：
10.1021/bi9719962
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发表时间：
1998-01-20
期刊：
影响因子：
2.9
通讯作者：
Bhagat, S
中科院分区：
文献类型：
--
作者：
Senior, AE;Bhagat, S
P-糖蛋白（P-Glycoprotein，Pgp）（又称多药耐药蛋白）含有两个核苷酸结合位点，均为ATP酶催化位点。共价试剂7-氯-4-硝基苯并-2-氧杂-1，3-二唑（NBD-Cl）在催化位点反应，并且在反应化学计量为1 mol NBD-Cl/mol Pgp时发生ATP酶活性的完全失活。我们表明，在反应化学计量的II摩尔/摩尔，这两个核苷酸位点成为标记在相对非选择性的方式。因此，两个核苷酸位点之间存在强相互作用，因为（a）一个位点与NBD-Cl的反应严重阻碍试剂与另一个位点的反应，和（B）一个位点的反应抑制稳态ATP酶，即两个位点都被抑制。钒酸盐捕获实验表明，当一个核苷酸位点与NBD-Cl反应时，在另一个完整的核苷酸位点中甚至不会发生单个ATP酶周转事件。因此，数据表明，Pgp中的两个核苷酸位点之间的催化协同性是非常强的，并且对于催化是强制性的。
P-Glycoprotein (Pgp) (also known as multidrug-resistance protein) contains two nucleotide binding sites, both of which are catalytic ATPase sites. The covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reacts in catalytic sites, and full inactivation of ATPase activity occurs at a reaction stoichiometry of 1 mol of NBD-Cl/mol of Pgp. We show that, at reaction stoichiometry of II mol/mol, both nucleotide sites become labeled in relatively nonselective fashion. There is therefore strong interaction between the two nucleotide sites because (a) reaction of one site with NBD-Cl severely impedes reaction of reagent with the other site, and (b) reaction of one site inhibits steady-state ATPase, i.e. both sites are inhibited. Vanadate-trapping experiments revealed that when one nucleotide site was reacted with NBD-Cl, not even a single ATPase turnover event could occur in the other, intact, nucleotide site. The data demonstrate therefore that catalytic cooperativity between the two nucleotide sites in Pgp is extremely strong and mandatory for catalysis.