Identification of the two histidine residues responsible for the inhibition by malonyl-CoA in peroxisomal carnitine octanoyltransferase from rat liver.
Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.
Morillas
M
M
Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy, Diagonal 643, 08028, Barcelona, Spain.
Clotet
J
J
Rubí
B
B
Serra
D
D
Asins
G
G
Ariño
J
J
Hegardt
F G
FG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002333:Carnitine Acyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D020675:Peroxisomes / Q000201:enzymology; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006639:Histidine; D008316:Malonyl Coenzyme A; D002333:Carnitine Acyltransferases; C030162:carnitine octanoyltransferase