A Single Amino Acid Change (Substitution of Glutamate 3 with Alanine) in the N-terminal Region of Rat Liver Carnitine Palmitoyltransferase I Abolishes Malonyl-CoA Inhibition and High Affinity Binding

Jianying Shi,Hongfa Zhu,Dennis N Arvidson,Gebre Woldegiorgis

doi:10.1074/jbc.274.14.9421

Abstract

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

Highlights

Carnitine palmitoyltransferase I (CPTI)1 catalyzes the conversion of long chain acyl-CoA to acylcarnitines in the presence of L-carnitine, the first reaction in the transport of long chain fatty acids from the cytoplasm to the mitochondria, a ratelimiting step in ␤-oxidation [1, 2]
Lar mechanism of the regulation of CPTI by malonyl-CoA is important in the design of drugs for control of excessive fatty acid oxidation in diabetes mellitus [10] and in myocardial ischemia where accumulation of acylcarnitines has been associated with arrhythmias [11]
P. pastoris was chosen as an expression system for L-CPTI and the mutants, because it does not have endogenous CPT activity [6, 12,13,14]

Summary

Introduction

Carnitine palmitoyltransferase I (CPTI)1 catalyzes the conversion of long chain acyl-CoA to acylcarnitines in the presence of L-carnitine, the first reaction in the transport of long chain fatty acids from the cytoplasm to the mitochondria, a ratelimiting step in ␤-oxidation [1, 2]. Mitochondria were isolated from the yeast strains separately expressing L-CPTI, and the deletion and point mutants were assayed for CPT activity, malonyl-CoA sensitivity, and binding, as described under “Experimental Procedures.” The IC50 is the concentration of malonyl-CoA needed to inhibit 50% of the activity of the yeast-expressed L-CPTI, and the results are the means Ϯ S.D. of at least three independent experiments with different mitochondrial preparations.

Results

Conclusion