A single amino acid change (substitution of the conserved Glu-590 with alanine) in the C-terminal domain of rat liver carnitine palmitoyltransferase I increases its malonyl-CoA sensitivity close to that observed with the muscle isoform of the enzyme.
Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the highly conserved C-terminal glutamate residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the glutamate residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9- to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged residues (alanine and glutamine) and/or a basic positively charged residue (lysine) significantly increased L-CPTI malonyl-CoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme.
Napal
Laura
L
Department of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, Beaverton, Oregon 97006-8921, USA.
Dai
Jia
J
Treber
Michelle
M
Haro
Diego
D
Marrero
Pedro F
PF
Woldegiorgis
Gebre
G
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002331:Carnitine / Q000737:chemistry / Q000494:pharmacology; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry*; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000737:chemistry; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009154:Mutation; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D010843:Pichia / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013552:Swine
D020033:Protein Isoforms; D005973:Glutamine; D010171:Palmitoyl Coenzyme A; D018698:Glutamic Acid; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D008239:Lysine; D000409:Alanine; D002331:Carnitine