An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C papaya. Based upon EST sequences, a full-length gene was identified in the genome of C papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser~(35)-Asp~(307)-His~(310)) with the nudeophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.
展開▼
機譯：在用兩性離子去污劑萃取并超聲處理之后，從凝膠木瓜膠乳中獲得對三丁酸甘油酯和短鏈乙烯基酯具有高比活性的酯酶（CpEst），然后進行凝膠過濾層析。盡管由于其存在于高分子量聚集體中而不能純化出完全同質的蛋白，但通過SDS-PAGE獲得了表觀分子量為41 kDa的主要蛋白帶。用胰蛋白酶消化該物質，并通過LC / ESI / MS / MS確定胰蛋白酶肽的氨基酸序列。這些序列用于從木瓜的表達序列標簽（EST）中鑒定部分cDNA（679 bp）?；贓ST序列，在番木瓜基因組中鑒定出全長基因，其具有1029bp的開放閱讀框，其編碼343個氨基酸殘基的蛋白質，理論分子量為38kDa。通過序列分析，將CpEst鑒定為屬于SGNH蛋白家族的GDSL基序羧酸酯水解酶，并鑒定出四個潛在的N-糖基化位點。推定的催化三聯體被定位（Ser?（35）-Asp?（307）-His?（310）），親核絲氨酸是GDSL基序的一部分。根據已知的X射線結構和序列比對建立CpEst的3D模型，發現催化三聯體暴露在分子表面，因此證實了四氫脂肪抑制素對CpEst抑制的結果表明該抑制劑可直接與活動站點。
展開▼
){kind=link}