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Originally published In Press as doi:10.1074/jbc.M007722200 on August 31, 2000

J. Biol. Chem., Vol. 275, Issue 49, 38410-38416, December 8, 2000
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Identification of Positive and Negative Determinants of Malonyl-CoA Sensitivity and Carnitine Affinity within the Amino Termini of Rat Liver- and Muscle-type Carnitine Palmitoyltransferase I*

Vicky N. Jackson, Victor A. ZammitDagger , and Nigel T. Price

From the Department of Cell Biochemistry, Hannah Research Institute, Ayr, Scotland KA6 5HL, United Kingdom

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 right-arrow 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 K0.5 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 Km 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.

* This work was supported by Diabetes UK, the British Heart Foundation, and the Scottish Executive.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Tel.: 44-0-1292-674058; Fax: 44-0-1292-674059; E-mail: zammitv@hri.sari.ac.uk.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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