Mutational analysis of the K+-competitive inhibitor site of gastric H,K-ATPase.
Mutational analysis of the K+-competitive inhibitor site of gastric H,K-ATPase.
复制标题
胃H,K-ATP酶K竞争性抑制剂位点的突变分析。
作者：
Vagin,O;Munson,K;Lambrecht,N;Karlish,SJ;Sachs,G
质子化咪唑并[1，2 α]吡啶（例如，SCH 28080）。识别的氨基酸在膜结构域中，影响SCH 28080抑制应该提供一个模板，用于建模一个luminally定向前庭在这种酶的晶体结构的基础上的SR钙-ATP酶。对H，K-ATP酶中5个保守的羧基残基Glu 343、Glu 795、Glu 820、Asp 824、Glu 936和独特的Lys 791进行了突变，并测定了突变对SCH 28080、Vmax和Km，app[NH 4 +]的影响。ATP水解数据的动力学分析表明，所有这些残基显着影响NH 4+离子与蛋白质的相互作用，但只有其中三个，Glu 795，Glu 936，和Lys 791，极大地影响SCH 28080抑制。Glu 795 Asp突变使Ki从64 ± 11增加到700 ± 110 nM。然而，由于突变Glu 795 Gln没有改变Ki（86 ± 31 nM），因此该位点对抑制剂动力学具有显著的空间效应。一个Glu 936 Asp突变导致非竞争性动力学，而谷氨酰胺取代没有影响无论是对抑制剂的亲和力或动力学的性质，这表明Glu 936侧链的长度是至关重要的独家结合的离子和SCH 28080。将Lys 791突变为Ser（存在于SCH 28080不敏感的Na，K-ATP酶中的残基）导致SCH 28080亲和力降低20倍，表明该残基在H，K-ATP酶相对于Na，K-ATP酶的SCH 28080选择性中起重要作用。Asp 824、Glu 343和Glu 820的突变使Ki增加了2 −3倍，这意味着这些残基在SCH 28080抑制中的作用相对较小。似乎SCH 28080的咪唑并吡啶部分在质子化状态下与腔侧空离子位点的带负电荷残基附近的残基（TM 4、-5、-6和-8）相互作用，而疏水苯环与TM 1或TM 2相互作用（后一结论基于来自光亲和标记的先前数据）。SCH 28080结合位点的完整性取决于H，K-ATP酶中Lys 791、Glu 936和Glu 795的存在。该区域的计算机生成的模型说明了先前显示影响SCH 28080抑制的残基（Cys 813，Ile 816，Thr 823，Met 334，Val 337）的可能参与，并且可以预测在泵的E2构象中排列SCH 28080结合前庭的其他残基。
The gastric H,K-ATPase is inhibited selectively and K+-competitively from its luminal surface by protonated imidazo[1,2α]pyridines (e.g., SCH28080). Identification of the amino acids in the membrane domain that affect SCH28080 inhibition should provide a template for modeling a luminally directed vestibule in this enzyme, based on the crystal structure of the sr Ca-ATPase. Five conserved carboxylic residues, Glu343, Glu795, Glu820, Asp824, Glu936, and unique Lys791 in the H,K-ATPase were mutated, and the effects of mutations on theKifor SCH28080,Vmax, andKm,app[NH4+] were measured. A kinetic analysis of the ATP hydrolysis data indicated that all of these residues significantly affect the interaction of NH4+ions with the protein but only three of them, Glu795, Glu936, and Lys791, greatly affected SCH28080 inhibition. A Glu795Asp mutation increased theKifrom 64 ± 11 to 700 ± 110 nM. Since, however, the mutation Glu795Gln did not change theKi(86 ± 31 nM), this site has a significant spatial effect on inhibitor kinetics. A Glu936Asp mutation resulted in noncompetitive kinetics while Gln substitution had no effect either on inhibitor affinity or on the nature of the kinetics, suggesting that the length of the Glu936 side chain is critical for the exclusive binding of the ion and SCH28080. Mutation of Lys791 to Ser, the residue present in the SCH28080-insensitive Na,K-ATPase, resulted in a 20-fold decrease in SCH28080 affinity, suggesting an important role of this residue in SCH28080 selectivity of the H,K-ATPase versus Na,K-ATPase. Mutations of Asp824, Glu343, and Glu820 increased theKi2−3-fold, implying a relatively minor role for these residues in SCH28080 inhibition. It appears that the imidazopyridine moiety of SCH28080 in the protonated state interacts with residues near the negatively charged residues of the empty ion site from the luminal side (TM4, -5, -6, and -8) while the hydrophobic phenyl ring interacts with TM1 or TM2 (the latter conclusion based on previous data from photoaffinity labeling). The integrity of the SCH28080 binding site depends on the presence of Lys791, Glu936, and Glu795 in H,K-ATPase. A computer-generated model of this region illustrates the possible involvement of the residues previously shown to affect SCH28080 inhibition (Cys813, Ile816, Thr823, Met334, Val337) and may predict other residues that line the SCH28080 binding vestibule in the E2conformation of the pump.