Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase (T123I and N228K).
Two naturally occurring mutants of human lecithin-cholesterol acyltransferase (LCAT), T123I and N228K, were expressed in COS-1 and Chinese hamster ovary cells, overproduced, and purified to homogeneity in order to study the structural and functional defects that lead to the LCAT deficiency phenotypes of these mutations. The mutants were expressed and secreted by transfected cells normally and had molecular weights and levels of glycosylation similar to wild type LCAT. The purified proteins (>98% purity) had almost indistinguishable structures and stabilities as determined by CD and fluorescence spectroscopy. Enzymatic activities and kinetic analysis of the pure enzyme 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 mutants 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 mutants with rHDL, the equilibrium binding constants were measured directly with isothermal titration calorimetry and surface plasmon resonance (SPR) methods. The results indicated that the affinities of the mutants for rHDL were only about 2-fold lower than the affinity of wild type LCAT (Kd = 2.3 x 10(-7) M). Together, the activity and equilibrium binding results suggest that 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 method indicate that normal LCAT dissociates from rHDL, on average, after one catalytic cycle.
Adimoolam
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S
Department of Biochemistry, University of Illinois College of Medicine at Urbana-Champaign, Urbana, Illinois 61801, USA.
Jin
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L
Grabbe
E
E
Shieh
J J
JJ
Jonas
A
A
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance
D017931:DNA Primers; D007862:Phosphatidylcholine-Sterol O-Acyltransferase