Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M-1s-1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E-64, solved at 1.73 and 1.98 Å, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E-64 and ananain explains the high inhibitory efficiency of E-64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

中文翻译：

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凤梨苷的晶体结构和底物特异性的测定。

菠萝蛋白酶（EC 3.4.22.31）占菠萝菠萝粗提物菠萝蛋白酶中总酶的不到10％，却产生了菠萝蛋白酶的大部分蛋白水解活性。尽管凤梨蛋白酶和茎菠萝蛋白酶（最丰富的菠萝蛋白酶半胱氨酸蛋白酶）之间具有高度的序列同一性，但与茎菠萝蛋白酶相比，凤梨蛋白酶显示出独特的化学性质，底物偏好和抑制特性。使用三肽底物文库（REPLi）进一步表征凤梨苷的底物特异性，并确定了由凤梨苷裂解的最佳底物。最佳的三肽PLQ产生的高kcat / Km值为1.7 x 106 M-1s-1，在Gln残基后确认发生了切割。未结合的凤梨苷的晶体结构以及凤梨苷和E-64的抑制复合物，在1.73和1.98Å处分辨，分别揭示了凤梨的几何平坦和开放的S1子位点。该亚位点可容纳各种P1底物残基，而窄而深的疏水性口袋状S2亚位点可容纳非极性P2残基，例如在特异性研究中观察到的优选Leu残基。E-64和凤梨苷之间原子相互作用的进一步说明说明了E-64对凤梨苷的高抑制效率。这些数据揭示了首个有关凤梨苷的深度结构和功能数据，并为进一步研究该酶的天然特性提供了基础。例如在特异性研究中观察到的优选Leu残基。E-64和凤梨苷之间原子相互作用的进一步说明说明了E-64对凤梨苷的高抑制效率。这些数据揭示了首个有关凤梨苷的深度结构和功能数据，并为进一步研究该酶的天然特性提供了基础。例如在特异性研究中观察到的优选Leu残基。E-64和凤梨苷之间原子相互作用的进一步说明说明了E-64对凤梨苷的高抑制效率。这些数据揭示了首个有关凤梨苷的深度结构和功能数据，并为进一步研究该酶的天然特性提供了基础。