Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase (T123I and N228K)

Two naturally occurring mutants of human lecithin-cholesterol acyltransfera se (LCAT), T123I and N228K, were expressed in COS-1 and Chinese hamster ova ry cells, overproduced, and purified to homogeneity in order to study the s tructural and functional defects that lead to the LCAT deficiency phenotype s of these mutations. The mutants were expressed and secreted by transfecte d cells normally and had molecular weights and levels of glycosylation simi lar to wild type LCAT, The purified proteins (>98% purity) had almost indis tinguishable structures and stabilities as determined by CD and fluorescenc e spectroscopy, Enzymatic activities and kinetic analysis of the pure enzym e forms showed that wild type LCAT and both mutants were reactive with the water-soluble substrate, p-nitrophenyl butyrate, indicating the presence of an intact core active site and catalytic triad, Both the T123I and N228K m utants had markedly depressed reactivity with reconstituted HDL (rHDL), but T123I retained activity with low density lipoprotein. To determine whether defective binding to rHDL was responsible for the low activity of both mut ants with rHDL, the equilibrium binding constants were measured directly wi th isothermal titration calorimetry and surface plasmon resonance (SPR) met hods. The results indicated that the affinities of the mutants for rHDL wer e only about 2-fold lower than the affinity of wild type LCAT (K-d = 2.3 x 10(-7) M). Together, the activity and equilibrium binding results suggest t hat the T123I mutant is defective in activation by apolipoprotein A-I, and the N228K mutant has impaired binding of lipid substrate to the active site . In addition, the kinetic binding rate constants determined by the SPR met hod indicate that normal LCAT dissociates from rHDL, on average, after one catalytic cycle.