Rudenko G, Bonten E, d'Azzo A, Hol W G
Department of Biological Structure, School of Medicine, University of Washington, Seattle 98195-7742, USA.
Structure. 1995 Nov 15;3(11):1249-59. doi: 10.1016/s0969-2126(01)00260-x.
The human 'protective protein' (HPP) forms a multi-enzyme complex with beta-galactosidase and neuraminidase in the lysosomes, protecting these two glycosidases from degradation. In humans, deficiency of HPP leads to the lysosomal storage disease galactosialidosis. Proteolytic cleavage of the precursor form of HPP involves removal of a 2 kDa excision peptide and results in a carboxypeptidase activity. The physiological relevance of this activity is, as yet, unknown.
The crystal structure of the 108 kDa dimer of the precursor HPP has been elucidated by making extensive use of twofold density averaging. The monomer consists of a 'core' domain and a 'cap' domain. Comparison with the distantly related wheat serine carboxypeptidase dimer shows that the two subunits in the HPP dimer differ by 15 degrees in mutual orientation. Also, the helical subdomain forming part of the cap domains is very different. In addition, the HPP precursor cap domain contains a 'maturation' subdomain of 49 residues which fills the active-site cleft. Merely removing the 'excision' peptide located in the maturation subdomain does not render the catalytic triad solvent accessible.
The activation mechanism of HPP is unique among proteases with known structure. It differs from the serine proteases in that the active site is performed in the zymogen, but is blocked by a maturation subdomain. In contrast to the zinc metalloproteases and aspartic proteases, the chain segment physically rendering the catalytic triad solvent inaccessible in HPP is not cleaved off to form the active enzyme. The activation must be a multi-step process involving removal of the excision peptide and major conformational changes of the maturation subdomain, whereas the conformation of the enzymatic machinery is probably almost, or completely, unaffected.
人类“保护蛋白”（HPP）在溶酶体中与β-半乳糖苷酶和神经氨酸酶形成多酶复合物，保护这两种糖苷酶不被降解。在人类中，HPP缺乏会导致溶酶体贮积病半乳糖唾液酸贮积症。HPP前体形式的蛋白水解切割涉及去除一个2 kDa的切除肽，并产生羧肽酶活性。这种活性的生理相关性尚不清楚。
通过广泛使用二次密度平均法阐明了108 kDa HPP前体二聚体的晶体结构。单体由一个“核心”结构域和一个“帽”结构域组成。与远缘相关的小麦丝氨酸羧肽酶二聚体比较表明，HPP二聚体中的两个亚基在相互取向上相差15度。此外，构成帽结构域一部分的螺旋亚结构域也非常不同。另外，HPP前体帽结构域包含一个由49个残基组成的“成熟”亚结构域，该亚结构域填充了活性位点裂隙。仅仅去除位于成熟亚结构域中的“切除”肽并不能使催化三联体暴露于溶剂中。
HPP的激活机制在已知结构的蛋白酶中是独特的。它与丝氨酸蛋白酶的不同之处在于，活性位点在酶原中就已形成，但被一个成熟亚结构域阻断。与锌金属蛋白酶和天冬氨酸蛋白酶相反，在HPP中物理上使催化三联体无法暴露于溶剂的链段不会被切割掉以形成活性酶。激活必须是一个多步骤过程，涉及切除肽的去除和成熟亚结构域的主要构象变化，而酶促机制的构象可能几乎或完全不受影响。
