Binding to the Low-Density Lipoprotein Receptor Accelerates Futile Catalytic Cycling in PCSK9 and Raises the Equilibrium Level of Intramolecular Acylenzyme

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• 作者：Kieran F. Geoghegan* ; Lise R. Hoth ; Alison H. Varghese ; Wen Lin ; James G. Boyd ; Matthew C. Griffor
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：April 7, 2009
• 年：2009
• 卷：48
• 期：13
• 页码：2941-2949
• 全文大小：256K
• 年卷期：v.48,no.13(April 7, 2009)
• ISSN：1520-4995

文摘

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) on target cells and lowers the level of receptor by impeding its recycling. PCSK9 is self-processed to a complex of its prodomain and catalytic domain like a typical protein convertase, but it does not develop normal proteolytic activity. Instead, its propeptide remains complexed with the catalytic domain, and the C-terminal Gln152 of the prodomain occupies the active site like a substrate for peptide synthesis. To probe its latent catalytic activity, PCSK9 and its complex with the soluble LDLR extracellular domain were separately transferred into H₂¹⁸O, and time point samples were analyzed by peptide mapping with mass spectrometry to measure the rate and extent of incorporation of ¹⁸O into the Gln152 carboxylate. In free wild-type or D374Y mutant PCSK9, the t_1/2 for exchange of ¹⁸O for both oxygens was near 5 min. This slow process progressed to completion, with the distribution of oxygen isotopes in the Gln152 carboxylate finally matching that in solvent. In contrast, exchange reached its final state in <30 s in LDLR-complexed D374Y mutant PCSK9, but 40% of the molecules gave data indicating the presence of only one ¹⁸O atom in Gln152. With support from further experiments, this was attributed to hydrolysis of acylenzyme in H₂¹⁶O during preparations for digestion and indicated that PCSK9 complexed with LDLR contains 40% intramolecular acylenzyme at equilibrium. The synthetic EGF-A domain of LDLR induced similar effects as the full-length receptor. The data suggest the existence of distinct conformational states in free and receptor-bound PCSK9.