Inhibitor and ion binding sites on the gastric H,K-ATPase
Inhibitor and ion binding sites on the gastric H,K-ATPase
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DOI：
10.1021/bi047761p
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发表时间：
2005-04-12
期刊：
影响因子：
2.9
通讯作者：
Sachs, G
中科院分区：
文献类型：
--
作者：
Munson, K;Garcia, R;Sachs, G
胃H，K-ATP酶催化H+与K+的电中性交换，作为酶磷酸化和去磷酸化的函数，在E-1/E-1-P（离子位点在）和E2 P/E-2（离子位点在）构象之间的转变。在这里，我们提出了E-2-P构象的H，K-ATP酶的同源性建模，作为预测该酶与两种已知类型的特异性抑制剂相互作用的方法。所有已知的质子泵抑制剂PPI与可从管腔表面接近的半胱氨酸813形成二硫键。这允许将结合位点分配到邻近由部分TM 4和TM 5与TM 6之间的环包围的Cys 813的腔前庭。K+竞争性咪唑-1，2 α-吡啶也结合到E-2-P构象的腔表面，并且它们的结合排除PPI反应。这两类抑制剂的结合位点的重叠结合定点诱变和半胱氨酸交联的结果，允许初步分配的对接模式，这些可逆的化合物在一个位置接近Glu 795，占详细的结构/活性的关系，这些化合物。新的E-2-P模型能够通过这种P-2型ATP酶分配酸分泌的可能机制。在Sr Ca-ATP酶中通过晶体学鉴定的几个离子结合侧链在Na，K-和H，K-ATP酶中是保守的。处于这些中间位置的H，K-ATP酶用赖氨酸取代了参与Na，K-ATP酶中K+结合的丝氨酸。水合氢离子与E1和E2-P结合的分子模型预测在E1 P和E2 P的构象转变期间，Asp 824、Glu 820和Glu 795之间结合的水合氢离子被Lys 791的R-NH3+向外置换。在低pH值下，管腔K+结合的网站建议之间的羰基氧的第四膜跨度和羧基氧的Glu 795和Glu 820的非螺旋部分。预计K+结合的该位点使这些羧酸盐与Lys 791的-NH3+基团之间的氢键不稳定，从而允许Lys 791侧链返回到其E-1位置。
The gastric H,K-ATPase catalyzes electroneutral exchange of H+ for K+ as a function of enzyme phosphorylation and dephosphorylation during transition between E-1/E-1-P (ion site in) and E2P/E-2 (ion site out) conformations. Here we present homology modeling of the H,K-ATPase in the E-2-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors. All known proton pump inhibitors, PPIs, form a disulfide bond with cysteine 813 that is accessible from the luminal surface. This allows allocation of the binding site to a luminal vestibule adjacent to Cys813 enclosed by part of TM4 and the loop between TM5 and TM6. K+ competitive imidazo-1,2 alpha-pyridines also bind to the luminal surface of the E-2-P conformation, and their binding excludes PPI reaction. This overlap of the binding sites of the two classes of inhibitors combined with the results of site-directed mutagenesis and cysteine cross-linking allowed preliminary assignment of a docking mode for these reversible compounds in a position close to Glu795 that accounts for the detailed structure/activity relationships known for these compounds. The new E-2-P model is able to assign a possible mechanism for acid secretion by this P-2-type ATPase. Several ion binding side chains identified in the sr Ca-ATPase by crystallography are conserved in the Na,K- and H,K-ATPases. Poised in the middle of these, the H,K-ATPase substitutes lysine in place of a serine implicated in K+ binding in the Na,K-ATPase. Molecular models for hydronium binding to E, versus E-2-P Predict outward displacement of the hydronium bound between Asp824, Glu820, and Glu795 by the R-NH3+ of Lys791 during the conformational transition from E1P and E2P. The site for luminal K+ binding at low pH is proposed to be between carbonyl oxygens in the nonhelical part of the fourth membrane span and carboxyl oxygens of Glu795 and Glu820. This site of K+ binding is predicted to destabilize hydrogen bonds between these carboxylates and the -NH3+ group of Lys791, allowing the Lys791 side chain to return to its E-1 position.