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.
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.
Jackson
V N
VN
Department of Cell Biochemistry, Hannah Research Institute, Ayr, Scotland KA6 5HL, United Kingdom.
Zammit
V A
VA
Price
N T
NT
D000818:Animals; D002331:Carnitine / Q000378:metabolism*; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008316:Malonyl Coenzyme A / Q000494:pharmacology*; D018482:Muscle, Skeletal / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010843:Pichia; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion
D010446:Peptide Fragments; D011994:Recombinant Proteins; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine