Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.
Biochemical characterization of Plasmodium falciparum dipeptidyl aminopeptidase 1.
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DOI：
10.1016/j.molbiopara.2010.08.004
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发表时间：
2011-01
影响因子：
1.5
通讯作者：
Klemba, Michael
中科院分区：
文献类型：
--
作者：
Wang, Flora;Krai, Priscilla;Deu, Edgar;Bibb, Brittney;Lauritzen, Conni;Pedersen, John;Bogyo, Matthew;Klemba, Michael
二肽基氨基肽酶1 （DPAP1）是一种重要的食物液泡酶，被认为在红细胞疟原虫的血红蛋白分解代谢中起作用。在这里，我们研究了DPAP1的生化特性，并将其与人类组织蛋白酶c的生化特性进行了比较。为了便于DPAP1的表征，我们开发了一种纯化的重组DPAP1的生产方法，其特性与天然酶非常相似。与组织蛋白酶C一样，DPAP1是一种氯化物活化酶，在酸性pH值下最有效地催化酰胺键水解。DPAP1的单体四聚体结构不同于组织蛋白酶C的四聚体结构，这表明四聚体化是组织蛋白酶C特异性功能所必需的。利用位置扫描合成组合文库分析了DPAP1和组织蛋白酶C的S1和S2亚位偏好。S1的偏好与其他c1家族半胱氨酸肽酶相似。DPAP1和组织蛋白酶C的S2亚位都接受脂肪族疏水残基、脯氨酸和一些极性残基，产生不同的特异性谱。DPAP1有效地催化了几种荧光二肽底物的水解；然而，令人惊讶的是，具有p2 -苯丙氨酸残基的潜在底物反而是竞争性抑制剂。总之，我们的生化数据表明，DPAP1通过弥合食物液泡内肽酶和氨基肽酶活性之间的差距，加速了血红蛋白中氨基酸的产生。两种可逆的组织蛋白酶C抑制剂能有效抑制重组DPAP1和天然DPAP1，从而验证了重组DPAP1在未来抑制剂发现和表征中的应用。
Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.
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DOI：
10.1073/pnas.96.24.13703
发表时间：
1999-11-23
影响因子：
11.1
作者：
Bessette, PH;Åslund, F;Georgiou, G
通讯作者：
Georgiou, G
影响因子：
4.8
作者：
DOLENC, I;TURK, B;TURK, V
通讯作者：
TURK, V
影响因子：
4.8
作者：
Dalal, Seema;Klemba, Michael
通讯作者：
Klemba, Michael
影响因子：
2.9
作者：
Cigic, B;Dahl, SW;Pain, RH
通讯作者：
Pain, RH
影响因子：
2.7
作者：
Bondebjerg, Jon;Fuglsang, Henrik;Naerum, Lars
通讯作者：
Naerum, Lars