Use of six chimeric proteins to investigate the role of intramolecular interactions in determining the kinetics of carnitine palmitoyltransferase I isoforms

The two isoforms of carnitine palmitoyltransferase I (CPT I; muscle (M)- and liver (L)-type) of the mitochondrial outer membrane have distinct kinetic characteristics with respect to their affinity for one of the substrates (l-carnitine) and the inhibitor malonyl-CoA. Moreover, they differ markedly in their hysteretic behavior with respect to malonyl-CoA and in their response to changes in the in vivo metabolic state. However, the two proteins are 62% identical and have the same overall structure. Using liver mitochondria, we have previously shown that the protein is polytopic within the outer membrane, comprising a 46-residue cytosolic N-terminal sequence, two transmembrane segments (TM1 and TM2) separated by a 27-residue loop, and a large catalytic domain (also cytosolic) (Fraser, F., Corstorphine, C. G., and Zammit, V. A. (1997) Biochem. J. 323, 711-718). We have now conducted a systematic study on six chimeric proteins constructed from combinations of three linear segments of rat L- and M-CPT I and on the two parental proteins to elucidate the effects of altered intramolecular interactions on the kinetics of CPT activity. The three segments were (i) the cytosolic N-terminal domain plus TM1, (ii) the loop plus TM2, and (iii) the cytosolic catalytic C-terminal domain. The kinetic properties of the chimeric proteins expressed in Pichia pastoris were studied. We found that alterations in the combinations of the N-terminal plus TM1 and C-terminal domains as well as in the N terminus plus TM1/TM2 pairings resulted in changes in the K(m) values for carnitine and palmitoyl-CoA and the sensitivity to malonyl-CoA of the L-type catalytic domain. The changes in affinity for malonyl-CoA and palmitoyl-CoA occurred independently of changes in the affinity for carnitine. The kinetic characteristics of the M-type catalytic domain and, in particular, its malonyl-CoA sensitivity were much less susceptible to influence by exchange of the other two segments of the protein. The marked difference in the response of the two catalytic domains to changes in the N-terminal domain and TM combinations explains the previously observed differences in the response of L- and M-CPT I to altered physiological state in intact mitochondria and to modulation of altered lipid molecular order of the mitochondrial outer membrane in vivo and in vitro.