Identification of an amino acid in the ATP binding site of Na+/K(+)-ATPase after photochemical labeling with 8-azido-ATP.
Identification of an amino acid in the ATP binding site of Na+/K(+)-ATPase after photochemical labeling with 8-azido-ATP.
复制标题
用 8-叠氮基-ATP 进行光化学标记后，鉴定 Na /K( )-ATP 酶 ATP 结合位点中的氨基酸。
DOI：
10.1021/bi00180a006
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Farley,RA
中科院分区：
文献类型：
--
作者：
Tran,CM;Scheiner-Bobis,G;Schoner,W;Farley,RA
修订稿于 1994 年 1 月 28 日收到• 摘要：[a-32P]-8-N3-ATP、[2-3H]-8-N3-ATP 和非放射性 8-N3-ATP 已被用作狗肾 Na+/K+-ATP 酶 ATP 结合位点的光亲和探针。 8-N3-ATP 先前已被证明能够以高亲和力与 Na+/K+-ATP 酶结合，成为 Na+/K+-ATP 酶的底物，并在紫外线照射下使酶失活 [Scheiner-Bobis, G., & Schoner, W.(1985) Eur. J.生物化学。 152、739-746]。 8-N3-ATP 竞争性抑制 [2, 8-3H]-ATP 与 Na+/K+-ATP 酶的高亲和力结合，Kd 为 3.4/tM，与报道的 8-N3-ATP 与酶结合的 KD 为 3.1/tM 相当。 8-N3-ATP 对 ATP 水解的抑制程度与 8-N3-ATP 共价掺入 Na+/K+-ATP 酶的化学计量呈线性相关，最高可抑制约 50% 的活性；然而，该蛋白与8-N 3-ATP之间的连接不稳定，并且8-N3-ATP的最大掺入量小于该蛋白的核苷酸结合能力。紫外光光解后，8-N3-ATP特异性地掺入到KC1存在下有限胰蛋白酶消化产生的Na+/K+-ATP酶α亚基的羧基端58-kDa片段中，且/3-亚基未被标记。用胰蛋白酶消化 8-N3-ATP 标记的 Na+/K+-ATP 酶，在对消化物进行 HPLC 分级分离后，鉴定出含有核苷酸的单峰。该峰中的肽经过纯化和测序，发现其氨基酸序列为 Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys-Tyr-Gln-Leu-Ser-Ile-His-Lys-Asn-Pro-Asn-Thr-Ser-Glu-Pro-Arg。该序列对应于 Na+/K+-ATPase a 亚基的氨基酸 470-495，并且在其他 P 型离子泵中也高度保守。与未标记肽的相同序列相比，光化学标记肽的序列中对应于赖氨酸480的第11轮测序仪的产率显着降低。这些结果表明，2-N3-ATP 从蛋白质的 ATP 结合位点内标记 Na+/K+-ATPase 的赖氨酸 480。Na+/K+-ATPase* (EC 3.6. 1.37) 是一种内在的膜嵌入酶，可逆电化学梯度跨细胞膜转运 Na+ 和 K+ 离子（Skou & Esmann，1992；Glynn，1993）。该酶由催化 a 亚基 (Mr= 112 000) 和糖基化/3 亚基 (A/r= 35 000) 组成。 Na+/K+-ATPase α-亚基的氨基酸序列与其他离子易位中发现的类似大小的多肽具有 20-75% 的同一性
Revised Manuscript Received January 28, 1994• abstract:[a-32P]-8-N3-ATP,[2-3H]-8-N3-ATP, and non-radioactive 8-N3-ATP have been used as photoaffinity probes of the ATP binding site of dog kidneyNa+/K+-ATPase. 8-N3-ATP has previously been shown to bind to Na+/K+-ATPase with high affinity, to be a substrate for Na+/K+-ATPase, and to inactivate the enzyme upon ultraviolet irradiation [Scheiner-Bobis, G., & Schoner, W.(1985) Eur. J. Biochem. 152, 739-746]. 8-N3-ATP competitively inhibits the high-affinity binding of [2, 8-3H]-ATP to Na+/K+-ATPase with a K, of 3.4/tM, which is comparable to the reported KD of 3.1/tM for the binding of 8-N3-ATP to the enzyme. The extent of inhibition of ATP hydrolysis by 8-N3-ATP was linearly correlated with the stoichiometry of covalent incorporation of 8-N3-ATP into Na+/K+-ATPase up to about 50% inhibition of activity; however, the linkage between the protein and 8-N 3-ATP was unstable, and the maximum incorporation of 8-N3-ATP was less than the nucleotide binding capacity of the protein. After photolysis with ultraviolet light, 8-N3-ATP was specifically incorporated into the carboxy-terminal 58-kDa fragment of the a-subunit of Na+/K+-ATPase generated by limited trypsin digestion in the presence of KC1, and the/3-subunit was not labeled. 8-N3-ATP-labeled Na+/K+-ATPase was digested with trypsin, and a single peak containing the nucleotide was identified after HPLC fractionation of the digest. The peptide in this peak was purified and sequenced and was found to have the amino acid sequence, Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys-Tyr-Gln-Leu-Ser-Ile-His-Lys-Asn-Pro-Asn-Thr-Ser-Glu-Pro-Arg. Thissequence corresponds to amino acids 470-495 of the Na+/K+-ATPase a-subunit and is also highly conserved among other P-type ion pumps. The yield from thesequencer at cycle 11, corresponding to lysine 480, was substantially reduced in the sequence of the photochemically labeled peptide compared to the same sequence of an unlabeled peptide. These results indicatethat 2-N3-ATP labels lysine 480 of Na+/K+-ATPase from within the ATP binding site of the protein.Na+/K+-ATPase*(EC 3.6. 1.37) is an intrinsic membrane-embedded enzyme that transports Na+ and K+ ions across cell membranes against their electrochemical gradients (Skou & Esmann, 1992; Glynn, 1993). The enzyme consists of a catalytic a-subunit (Mr= 112 000) and a glycosylated/3-subunit (A/r= 35 000). The amino acid sequence of the a-subunit of Na+/K+-ATPase has 20-75% identity with polypeptides of similar size found in other ion-translocating