Substitution of glutamate-3, valine-19, leucine-23, and serine-24 with alanine in the N-terminal region of human heart muscle carnitine palmitoyltransferase I abolishes malonyl CoA inhibition and binding.
The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.
Zhu
Hongfa
H
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, 20000 N.W. Walker Road, Beaverton 97006-8921, USA.
Shi
Jianying
J
Treber
Michelle
M
Dai
Jia
J
Arvidson
Dennis N
DN
Woldegiorgis
Gebre
G
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002334:Carnitine O-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D008316:Malonyl Coenzyme A; D004247:DNA; D002334:Carnitine O-Palmitoyltransferase