Binding to the low-density lipoprotein receptor accelerates futile catalytic cycling in PCSK9 and raises the equilibrium level of intramolecular acylenzyme.

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 Gin 152 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 H(2)(18)O, and time point samples were analyzed by peptide mapping with mass spectrometry to measure the rate and extent of incorporation of (18)O into the Gln152 carboxylate. In free wild-type or D374Y mutant PCSK9, the t(1/2) for exchange of (18)O 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 similar to 40% of the molecules gave data indicating the presence of only one (18)O atom in Gin 152. With support from further experiments, this was attributed to hydrolysis of acylenzyme in H(2)(16)O during preparations for digestion and indicated that PCSK9 complexed with LDLR contains similar to 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.