ROLE OF THE ARG(123)-TYR(166) PAIRED HELIX OF APOLIPOPROTEIN-A-I IN LECITHIN-CHOLESTEROL ACYLTRANSFERASE ACTIVATION

The Arg(123)-Tyr(166) central and Ala(190)-Gln(243) carboxy-terminal p airs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Delta(Arg(123)-Tyr(166)),del A-II(Se r(12)-Ala(75))) and apoA-I(Delta(Ala(190)-Gln(243)),del A-II(Ser(12)-G ln(77))) chimeras, respectively, The structures of these chimeras in a queous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation propert ies of the rHDL were studied, Recombinant human apoA-I and the chimera s were expressed in Escherichia coli and purified from the periplasmic space, Binding of the apolipoproteins with palmitoyloleoylphosphatidy lcholine was associated with a similar shift of Trp fluorescence maxim a from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, resp ectively, All rHDL had a Stokes radius of 4.8 nm and contained 2 apoli poprotein molecules/particle. Circular dichroism measurements revealed eight a-helices per apoA-I and per chimera molecule, The catalytic ef ficiencies of LCAT activation were 1.5 +/- 0.33 (mean +/- S.D.; n = 3) , 0.054 +/- 0.009 (p < 0.001 versus apoA-I), and 1.3 +/- 0.32 (p = not significant versus apoA-I) nmol of cholesteryl ester/h/mu M, respecti vely. The lower LCAT activity of the central domain chimera was due to a 27-fold reduced V-max with unaltered K-m, Binding of radiolabeled L CAT to rHDL of apoA-I and apoA-I(Delta(Arg(123)-Tyr(166)),del A-II(Ser (12)-Ala(75))) was very similar. In conclusion, although substitution of the Arg(123)-Tyr(166) central or Ala(190)-Gln(243) carboxyl-termina l pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I r educes the rate of LCAT activity that is independent of binding to rHD L.