Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31

BACKGROUND: Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in Familial LCAT Deficiency (FLD), an autosomal recessive disorder characterized by low HDL, anemia and renal disease. OBJECTIVE: To investigate the mechanism by which Compound A, a small heterocyclic amine, activates LCAT. METHODS: Effect of Compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and NMR was used to determine Compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into Compound A effects on LCAT structure and activity. RESULTS: Compound A increased LCAT activity in a subset (3 out of 9) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by Compound A. Substitution of Cys31 with charged residues (Glu, Arg and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe and Met) increased activity up to 3-fold (p<0.005). Mass spectrometry of a tryptic digest of LCAT incubated with Compound A revealed a +103.017 m/z adduct on Cys31, consistent with addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of Compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by Compound A were identified by testing Compound A derivatives. Finally, sulfhydryl-reactive β-lactams were developed as a new class of LCAT activators. CONCLUSIONS: Compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.