A single amino acid change (substitution of glutamate 3 with alanine) in the N-terminal region of rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA inhibition and high affinity binding.
We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.
Shi
J
J
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000, USA.
Zhu
H
H
Arvidson
D N
DN
Woldegiorgis
G
G
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017354:Point Mutation; D051381:Rats; D013329:Structure-Activity Relationship
D018698:Glutamic Acid; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D000409:Alanine