Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase I

Elucidation of the membrane topology of carnitine palmitoyltransferase (CPT ) I showed that the extreme N-terminus is involved in determining the sensi tivity of the liver (L) isoform to malonyl-CoA and suggested that interacti on between the two cytosolic segments of the CPT I molecule determines the kinetic characteristics of the enzyme. Work with chimaeric liver/muscle-iso form (L/M) proteins constructed from all six possible combinations of three domains [N-terminus plus transmembrane domain 1 (TM1), loop plus TM2 and C -domain] expressed in Pichia pastoris showed that the precise N-C and TM1-T M2 pairings determine the overall kinetic parameters of the protein. Discre te short sequences within the respective N-terminal regions have negative o r positive effects on malonyl-CoA sensitivity (L-isoform) or the K-m for ca rnitine (M-isoform) in the full-length proteins, thus imparting to them the ir distinctive kinetic characteristics. Interactions within N-terminal doma ins also seem to be important in the targeting of the protein to microsomes in the P. pastoris expression system.