Identification by mutagenesis of a conserved glutamate (Glu487) residue important for catalytic activity in rat liver carnitine palmitoyltransferase II.
Mammalian mitochondrial membranes express two active but distinct carnitine palmitoyltransferases: carnitine palmitoyltransferase I (CPTI), which is malonyl coA-sensitive and detergent-labile; and carnitine palmitoyltransferase II (CPTII), which is malonyl coA-insensitive and detergent-stable. To determine the role of the highly conserved C-terminal acidic residues glutamate 487 (Glu(487)) and glutamate 500 (Glu(500)) on catalytic activity in rat liver CPTII, we separately mutated these residues to alanine, aspartate, or lysine, and the effect of the mutations on CPTII activity was determined in the Escherichia coli-expressed mutants. Substitution of Glu(487) with alanine, aspartate, or lysine resulted in almost complete loss in CPTII activity. Because a conservative substitution mutation of this residue, Glu(487) with aspartate (E487D), resulted in a 97% loss in activity, we predicted that Glu(487) would be at the active-site pocket of CPTII. The substantial loss in CPTII activity observed with the E487K mutant, along with the previously reported loss in activity observed in a child with a CPTII deficiency disease, establishes that Glu(487) is crucial for maintaining the configuration of the liver isoform of the CPTII active site. Substitution of the conserved Glu(500) in CPTII with alanine or aspartate reduced the V(max) for both substrates, suggesting that Glu(500) may be important in stabilization of the enzyme-substrate complex. A conservative substitution of Glu(500) to aspartate resulted in a significant decrease in the V(max) for the substrates. Thus, Glu(500) may play a role in substrate binding and catalysis. Our site-directed mutagenesis studies demonstrate that Glu(487) in the liver isoform of CPTII is essential for catalysis.
Zheng
Guolu
G
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton 97006-8921, USA.
Dai
Jia
J
Woldegiorgis
Gebre
G
D000595:Amino Acid Sequence; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005973:Glutamine; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D005973:Glutamine; D002334:Carnitine O-Palmitoyltransferase