Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages.
Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages.
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DOI：
10.1021/acs.biochem.5b01157
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发表时间：
2016-02-16
期刊：
影响因子：
2.9
通讯作者：
Stone E
中科院分区：
文献类型：
--
作者：
Li W;Irani S;Crutchfield A;Hodge K;Matthews W;Patel P;Zhang YJ;Stone E
人天冬酰胺酶样蛋白1 （hASRGL1）是n端亲核蛋白（Ntn）家族的成员，可水解l-天冬酰胺和异天冬氨酸二肽。新生蛋白折叠成αβ -βα夹心折叠同源二聚体，在G167-T168键上切割自己的肽主链，导致酶的活性形式。然而，由于G167-T168肽键的分子内裂解仅完成≤50%，因此对hASRGL1的生物物理研究很困难。我们利用我们之前的观察，即分子内加工增加热稳定性，并开发了一种差分扫描荧光测定法，首次允许直接检测不同的加工中间体。对这些中间体的动力学分析表明，hASRGL1亚基的一个亚基的裂解大大降低了相邻单体的加工速率，一项诱变研究表明，二聚体界面的稳定在这一过程中起着关键作用。我们还报道了对保守活性位点残基的全面分析，并描述了它们在自动加工和底物水解中的相对作用。除了先前报道的甘氨酸可以选择性地加速hASRGL1的裂解外，我们还发现了几种新的小分子激活剂，它们也可以促进分子内加工。结构-活性分析支持了多个带负电荷的小分子在hASRGL1的活性位点内相互作用作为促进裂解的碱基的假设。总的来说，我们的研究提供了对Ntn水解酶家族成员成熟过程的机制理解。
The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes L-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ–βα sandwich fold homodimer that cleaves its own peptide backbone at the G167–T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167–T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure–activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.