Identification by mutagenesis of a conserved glutamate (Glu487) residue important for catalytic activity in rat liver carnitine palmitoyltransferase II

Abstract

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 (Glu487) and glutamate 500 (Glu500) 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 Glu487 with alanine, aspartate, or lysine resulted in almost complete loss in CPTII activity. Because a conservative substitution mutation of this residue, Glu487 with aspartate (E487D), resulted in a 97% loss in activity, we predicted that Glu487 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 Glu487 is crucial for maintaining the configuration of the liver isoform of the CPTII active site. Substitution of the conserved Glu500 in CPTII with alanine or aspartate reduced the Vmax for both substrates, suggesting that Glu500 may be important in stabilization of the enzyme-substrate complex. A conservative substitution of Glu500 to aspartate resulted in a significant decrease in the Vmax for the substrates. Thus, Glu500 may play a role in substrate binding and catalysis. Our site-directed mutagenesis studies demonstrate that Glu487 in the liver isoform of CPTII is essential for catalysis.