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J. Biol. Chem., Vol. 275, Issue 26, 19707-19712, June 30, 2000

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Identification of a Sequence of Apolipoprotein A-I Associated with the Activation of Lecithin:Cholesterol Acyltransferase^*

Dmitri Sviridov^§, Anh Hoang, William H. Sawyer^¶, and Noel H. Fidge

From the  Baker Medical Research Institute, Melbourne 8008 and the ^¶ Department of Biochemistry, University of Melbourne, Parkville 3052, Victoria, Australia

We aimed to distinguish between the effects of mutations in apoA-I on the requirements for the secondary structure and a specific amino acid sequence for lecithin:cholesterol acyltransferase (LCAT) activation. Several mutants were constructed targeting region 140-150: (i) two mutations affecting -helical structure, deletion of amino acids 140-150 and substitution of Ala¹⁴³ for proline; (ii) two mutations not affecting -helical structure, substitution of Val¹⁴⁹ for arginine and substitution of amino acids 63-73 for sequence 140-150; and (iii) a mutation in a similar region away from the target area, deletion of amino acids 63-73. All mutations affecting region 140-150 resulted in a 4-42-fold reduction in LCAT activation. Three mutations, apoA-I(140-150), apoA-I(P143A), and apoA-I(140-150  63-73), affected both the apparent V_max and K_m, whereas the mutation apoA-I(R149V) affected only the V_max. The mutation apoA-I(63-73) caused only a 5-fold increase in the K_m. All mutants, except apoA-I(P143A) and apoA-I(63-73), were active in phospholipid binding assay. All mutants, except apoA-I(P143A), formed normal discoidal complexes with phospholipid. The mutation apoA-I(63-73) caused a significant reduction in the stability of apoA-I·phospholipid complexes in denaturation experiments. Combined, our results strongly suggest that although the correct conformation and orientation of apoA-I in the complex with lipids are crucial for activation of LCAT, when these conditions are fulfilled, activation also strongly depends on the sequence that includes amino acids 140-150.

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