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)- an d 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 respo nse to changes in the in vivo metabolic state. However, the two proteins ar e 62% identical and have the same overall structure. Using liver mitochondr ia, we have previously shown that the protein is polytopic within the outer membrane, comprising a 46-residue cytosolic N-terminal sequence, two trans membrane segments (TM1 and TM2) separated by a 27-residue loop, and a large catalytic domain (also cytosolic) (Fraser, F., Corstorphine, C. G., and Za mmit, V. A. (1997) Biochem. J. 323, 711-718). We have now conducted a syste matic 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 kinetic s of CPT activity. The three segments were (i) the cytosolic N-terminal dom ain plus TM1, (ii) the loop plus TM2, and (iii) the cytosolic catalytic C-t erminal domain. The kinetic properties of the chimeric proteins expressed i n Pichia pastoris were studied. We found that alterations in the combinatio ns of the N-terminal plus TM1 and C-terminal domains as well as in the N te rminus plus TM1/TM2 pairings resulted in changes in the K-m values for carn itine and palmitoyl-CoA and the sensitivity to malonyl-CoA of the L-type ca talytic domain. The changes in affinity for malonyl-CoA and palmitoyl-CoA o ccurred independently of changes in the affinity for carnitine, The kinetic characteristics of the M-type catalytic domain and, in particular, its mal onyl-CoA sensitivity were much less susceptible to influence by exchange of the other two segments of the protein. The marked difference in the respon se of the two catalytic domains to changes in the N-terminal domain and TM combinations explains the previously observed differences in the response o f L- and M-CPT I to altered physiological state in intact mitochondria and to modulation of altered lipid molecular order of the mitochondrial outer m embrane in vivo and in vitro.