The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron‐shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp‐62 was substituted with alanine, asparagine, or glutamate residue. The Asp‐62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp‐62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.

中文翻译：

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M42氨肽酶催化位点：严格保守的天冬氨酸残基的结构和功能作用。

M42氨基肽酶是广泛分布于原核生物中的双核氨基肽酶家族。它们潜在地与蛋白酶体相关，实现了肽的完全破坏。它们最独特的特征是它们的四级结构，即由十二个亚基组成的四面体形状的粒子。M42氨基肽酶的催化位点由七个保守残基定义。其中五个与金属离子结合有关，这对维持活性和低聚状态都很重要。第六个保守残基，谷氨酸，是在肽键水解过程中使水分子去质子化的催化碱基。第七残基是天冬氨酸，其功能尚不清楚。但是，该天冬氨酸残基必须起关键作用，因为它在所有MH家族酶中都严格保守。它与组氨酸残基和M2结合位点的金属离子形成某种催化三联体。我们评估了它在TmPep1050（一种M42氨基肽酶）中的作用maritima，通过一种突变方法。Asp-62被丙氨酸，天冬酰胺或谷氨酸残基取代。Asp-62取代完全废除了TmPep1050活性并阻止十二聚体形成。它们还干扰金属离子的结合，因为每个亚基只能结合一个钴离子，而不是两个。Asp62Ala变体的结构在1.5Å处解析，显示了取代如何影响活性位点折叠。我们提出了Asp-62的结构性作用，有助于稳定活性位点中的关键环，并正确定位M1位点的催化碱基和金属离子配体。