Specificity of the interactions between Glu-3, Ser-24, and Gln-30 within the N-terminal segment of rat liver mitochondrial overt carnitine palmitoyltransferase (L-CPT I) in determining the malonyl-CoA sensitivity of the enzyme

Using deletion mutants of rat liver-type carnitine palmitoyltransferase I ( L-CPT I) expressed in Pichia pastoris, two contiguous discrete sequences wi thin its N-terminal segment have been shown to be positive (residues 3-18) and negative (19-30) determinants, respectively, of the malonyl-CoA sensiti vity of the enzyme. The specific interactions among the three individual re sidues responsible for these opposing effects within these two regions are here investigated in the context of the full-length protein. The pro-inhibi tory effects are due to Glu-3 [Shi et al. (1999) J. Biol. Chem. 274, 9421-9 426]. We now find that Asp can only partially substitute for Glu-3, whereas the Glu-3Gln mutation has the same effect as the Glu-3Ala mutation. This s uggests that a negative charge in this position is essential and that the l onger side chain of glutamate is essential for optimal malonyl-CoA sensitiv ity. Residues within the predicted a-helical 19-30 region responsible for d ecreasing the sensitivity to malonyl-CoA are shown to be neither the three basic (Arg-22, His-25, and Lys-29) nor the two acidic (Asp-20 and Glu-26) r esidues, as their mutation to Ala produced only small positive effects on m alonyl-CoA sensitivity. The residues responsible were identified as Ser-24 and Gln-30, and their effect was shown to be entirely dependent on the pres ence of Glu-3. This result reveals that the major sensitization of L-CPT I to malonyl-CoA observed upon deletion of residues 19-30 is not due to a spa cer effect with respect to Glu-3 but rather the loss of the two specific re sidues now identified.