Mapping the active site of angiotensin-converting enzyme by transferred NOE spectroscopy
Mapping the active site of angiotensin-converting enzyme by transferred NOE spectroscopy
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DOI：
10.1021/jm990194x
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发表时间：
2000-06-01
影响因子：
7.3
通讯作者：
Meyer, B
中科院分区：
文献类型：
--
作者：
Mayer, M;Meyer, B
通过转移noesi实验研究了5种糠酰丙烯酰（fa）氨基酸衍生物fa- phe、fa- phe、fa- gly - leu - nh2、fa- ala - lys和fa- trp与分子量为130 kDa的血管紧张素转换酶（ACE）的相互作用。与ACE结合的fa衍生物的鉴定及其相对亲和力的测定可以直接从化合物混合物中完成。在5个fa衍生物中，我们发现fa- phe、fa- trp和fa- gly - leu - nh2与ACE的结合比其他两个更强。从核磁共振光谱中确定了fa-Phe的解离常数为5 × 10(-4) M.大量的二肽竞争性地从受体口袋中取代了fa-Trp和fa-Phe- phe，从而可以绘制结合位点。此外，添加二肽后，fa-Phe、fa-Ala-Lys和fa-Gly-Leu-NH2的相对亲和力发生了变化，其中fa-Gly-Leu-NH2的结合最强。此外，S1‘和S2’位点的强抑制剂卡托普利的存在导致fa-Phe的NOE转移强度相同，这表明它仅结合S1和S2亚位点。通过在转移NOE研究中使用大量过量配体，即使存在相对少量的蛋白质，也可以从混合物中快速筛选结合特异性。
The interaction of five furylacryloyl (fa)-amino acid derivatives, fa-Phe, fa-Phe-Phe, fa-Gly-Leu-NH2, fa-Ala-Lys, and fa-Trp, with angiotensin-converting enzyme (ACE), a protein of MW = 130 kDa, was studied by transferred NOESY experiments. Identification of fa derivatives binding to ACE as well as determination of their relative affinities could be accomplished directly from the compound mixtures. Of the five fa derivatives we found that fa-Phe, fa-Trp, and fa-Gly-Leu-NH2 bind more strongly to ACE than the other two. The dissociation constant of fa-Phe was determined from NMR spectra to 5 x 10(-4) M. A large excess of dipeptides competitively displaced fa-Trp and fa-Phe-Phe from the receptor pocket, allowing the binding site to be mapped. Also, the relative affinities of the fa-Phe, fa-Ala-Lys, and fa-Gly-Leu-NH2 changed after addition of the dipeptides with fa-Gly-Leu-NH2 showing the strongest binding. In addition, the presence of a strong inhibitor of the S1' and S2' sites, namely captopril, resulted in the same transferred NOE intensities of fa-Phe, indicating that it binds solely to the S1 and S2 subsites. A rapid screening of binding specificity from mixtures is possible by using a large excess of ligand(s) in transferred NOE studies, even when relatively small amounts of protein are present.