Abstract
The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonyl-CoA. Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 --> Ala) or Delta(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant Delta(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I. This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19-82 of the enzyme. This was confirmed in the construct Delta(19-30), which was found to be 50-fold more sensitive than wild-type L-CPT I. Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions. This behavior was dependent on the presence of Glu-3, with the mutant E3A-Delta(19-30) having kinetic characteristics similar to those of the E3A mutant. The increase in the sensitivity of the L-CPT I-Delta(19-30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K(0.5) for this substrate. Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from Delta(3-18) to Delta(1-80). However, deletion of residues 3-18 from M-CPT I affected the K(m) for carnitine of this isoform, but not of L-CPT I. These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme.
Highlights
It has been shown that the extreme amino terminus of the nascent L-carnitine palmitoyltransferase I (CPT I) is retained in the mature protein (6) and, that it is essential for the expression of malonyl-CoA sensitivity (5, 7)
We made preliminary observations on the ⌬(1– 82) mutant expressed in P. pastoris, which suggested that, is the protein expressed by this construct sensitive to malonyl-CoA but it is highly significantly more so than the E3A mutant expressed in the same yeast expression system
The original concept (5), that the cytosolic amino-terminal segment is essential for the expression of malonyl-CoA sensitivity, applies to the native L-CPT I, it is obvious from the present data that this overall effect results from a far more complex interaction than has hitherto been envisaged, between positive and negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of the protein
Summary
L-CPT I, liver isoform of carnitine palmitoyltransferase; M-CPT I, muscle isoform; TM, transmembrane segment; IC50, concentration of malonyl-CoA that gives 50% inhibition of CPT activity; K0.5, concentration of palmitoyl-CoA that gives half the maximal activity of CPT observed at the carnitine concentration used in the assay; PCR, polymerase chain reaction. CPT I exists in two isoforms (Liver and Muscle), which have considerable sequence similarity but differ greatly, and inversely, in their malonyl-CoA sensitivity and Km for carnitine (3, 4) It is a polytopic protein, with two transmembrane (TM) segments and amino and carboxyl segments (approximately 46 and 652 residues, respectively) that are both exposed on the cytosolic aspect of the membrane (5). The precise amino-to-carboxyl pairings affected the sensitivity to malonyl-CoA and the Km for palmitoyl-CoA of the chimeric CPTs, whereas TM1-TM2 pairings affected the affinity for carnitine (11) These studies highlighted our long-standing observations on the importance of the interaction of CPT I with the membrane, of which it is an integral protein, for the expression of its kinetic characteristics (12, 13) and which more recently have been confirmed by work on the reconstitution of the solubilized or purified recombinant protein in vesicles of different lipid molecular order (14, 15). Of the many mutants of CPT I studied by various workers, these are the first to express a much more malonyl-CoA-sensitive enzyme
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