Kinetic, circular dichroism and fluorescence studies on heterologously expressed carnitine palmitoyltransferase II.
Km estimates for carnitine and palmitoyl-CoA of heterologously expressed rat liver carnitine palmitoyl-transferase-II (rCPT-II) were 950 +/- 27 microM and 34 +/- 6 microM, respectively. Vmax for the enzyme was 1.8 mumol/min/mg purified protein. Consistent with an ordered reaction mechanism in which palmitoyl-CoA binds first, SDZ CPI 975, a reversible carnitine palmitoyltransferase inhibitor containing both carnitine and alkyl moieties, inhibited rCPT-II competitively with carnitine and uncompetitively with palmitoyl-CoA. Substrate-enzyme interactions were examined by circular dichroism (CD) and fluorescence. Both carnitine and palmitoyl-CoA alone induced conformational changes in the enzyme; dissociation constant estimates by CD for carnitine and palmitoyl-CoA were 41 +/- 5 microM and 7 +/- 2 microM, respectively.
Mann
W R
WR
Metabolic Diseases Department, Sandoz Research Institute, Sandoz Pharmaceuticals Corp., East Hanover, New Jersey 07936-1080, USA.
Yan
B
B
Dragland
C J
CJ
Bell
P A
PA
D000818:Animals; D002331:Carnitine / Q000494:pharmacology; D002334:Carnitine O-Palmitoyltransferase / Q000187:drug effects / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D010171:Palmitoyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine