Identification of the active‐site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin

FEBS Letters

Hadváry, Paul¹  Märki, Hans Peter¹  Lüthi-Peng, Qiaoqian¹ 

[1] Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland

关键词: Human pancreatic lipase active-site serine;    Tetrahydrolipstatin (THL);    Structure of lipase-THL adduct;    CZE;    capillary zone electrophoresis;    FABMS;    fast atom bombardment mass spectrometry;    FHHA;    5-formylleucyloxy-2-hexyl-2-hexadecenoic acid;    hPL;    human pancreatic lipase;    pPL;    porcine pancreatic lipase;    TFA;    trifluoroacetic acid;    THL;    tetrahydrolipstatin;   

DOI  :  10.1016/0014-5793(92)80112-T

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [³H], [¹⁴C]tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the δ-lactone Ro 40–444] in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152…His-263…Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.

【 授权许可】

Unknown