KINETIC, CIRCULAR-DICHROISM AND FLUORESCENCE STUDIES ON HETEROLOGOUSLY EXPRESSED CARNITINE PALMITOYLTRANSFERASE-II

K-m estimates for carnitine and palmitoyl-CoA of heterologously expres sed rat liver carnitine palmitoyltransferase-II (rCPT-II) were 950+/-2 7 mu M and 34+/-6 mu M, respectively. V-max for the enzyme was 1.8 mu mol/min/mg purified protein. Consistent with an ordered reaction mecha nism in which palmitoyl-CoA binds first, SDZ CPI 975, a reversible car nitine palmitoyltransferase inhibitor containing both carnitine and al kyl moieties, inhibited rCPT-II competitively with carnitine and uncom petitively with palmitoyl-CoA. Substrate-enzyme interactions were exam ined by circular dichroism (CD) and fluorescence. Both carnitine and p almitoyl-CoA alone induced conformational changes in the enzyme; disso ciation constant estimates by CD for carnitine and palmitoyl-CoA were 41+/-5 mu M and 7+/-2 mu M, respectively.