Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme.
Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme.
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DOI：
10.1021/bi801192h
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发表时间：
2008-12-02
期刊：
影响因子：
2.9
通讯作者：
Tang, Wei-Jen
中科院分区：
文献类型：
--
作者：
Malito, Enrico;Ralat, Luis A.;Manolopoulou, Marika;Tsay, Julie L.;Wadlington, Natasha L.;Tang, Wei-Jen
胰岛素降解酶 (IDE) 利用大型催化室选择性地结合和降解肽底物，例如胰岛素和 β 淀粉样蛋白 (Aβ)。与底物的紧密相互作用发生在距催化中心约 30Å 的外部位点处，该外部位点锚定底物的 N 末端，以促进催化位点处的结合和随后的裂解。然而，IDE 也会降解太短而无法同时占据催化位点和外部位点的肽底物。在这里，我们使用激肽作为模型系统来研究短肽人类 IDE 的动力学和调节。 IDE 专门降解 Pro/Phe 位点的缓激肽和激肽。缓激肽结合 IDE 的 1.9Å 晶体结构揭示了缓激肽与外部位点的结合，而不是与催化位点的结合。与观察到的高 Km 值一致，这表明缓激肽对 IDE 的亲和力较低。该结构还为短肽在外部位点的结合如何调节底物识别提供了分子基础。我们还发现，生理相关浓度的 S-亚硝基化剂和氧化剂可有效抑制人 IDE。半胱氨酸定向修饰起着关键作用，因为缺乏所有 13 个半胱氨酸的 IDE 突变体对 S-亚硝基-谷胱甘肽、过氧化氢或 N-乙基马来酰亚胺的抑制不敏感。具体而言，人 IDE 的半胱氨酸 819 位于催化室内，指向延伸的疏水口袋，对于失活至关重要。该残基的硫醇定向修饰可能会导致局部结构扰动，从而减少底物结合和催化作用。
Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid β (Aβ). Tight interactions with substrates occur at an exosite located ~30Å away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9Å crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite, and not to the catalytic site. In agreement with observed high Km values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all thirteen cysteines is insensitive to the inhibition by S-nitroso-glutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing towards an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.
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影响因子：
3.9
作者：
ARNELLE, DR;STAMLER, JS
通讯作者：
STAMLER, JS
影响因子：
3.5
作者：
KUROCHKIN, IV;GOTO, S
通讯作者：
GOTO, S
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1107/s0907444902016657
发表时间：
2002-11-01
影响因子：
2.2
作者：
Adams, PD;Grosse-Kunstleve, RW;Terwilliger, TC
通讯作者：
Terwilliger, TC
DOI：
10.1083/jcb.109.3.1301
发表时间：
1989-09
期刊：
The Journal of cell biology
影响因子：
--
作者：
Garcia JV;Gehm BD;Rosner MR
通讯作者：
Rosner MR