Abstract
Muscle carnitine palmitoyltransferase II (CPT II) deficiency is associated with various mutations in CPT2 gene. In the present study, the impact of the two CPT II variants P50H and Y479F were characterized in terms of stability and activity in vitro in comparison to wildtype (WT) and the well investigated variant S113L. While the initial enzyme activity of all variants showed wild-type-like behavior, the activity half-lives of the variants at different temperatures were severely reduced. This finding was validated by the investigation of thermostability of the enzymes using nano differential scanning fluorimetry (nanoDSF). Further, it was studied whether the protein stabilizing diphosphatidylglycerol cardiolipin (CL) has an effect on the variants. CL indeed had a positive effect on the stability. This effect was strongest for WT and least pronounced for variant P50H. Additionally, CL improved the catalytic efficiency for CPT II WT and the investigated variants by twofold when carnitine was the varied substrate due to a decrease in KM. However, there was no influence detected for the variation of substrate palmitoyl-CoA. The functional consequences of the stabilization by CL in vivo remain open.
Highlights
The β-oxidation of long-chain fatty acids (LCFAs) is the major source of energy for skeletal muscle
The so-called carnitine shuttle consists of carnitine palmitoyltransferase I (CPT I), carnitine:acylcarnitine translocase (CACT) and carnitine palmitoyltransferase II (CPT II)
The enzyme assay was based on an adaptation of Rufer et al [30] for the transfer reaction of the palmitic acid from palmitoyl-CoA to L-carnitine
Summary
The β-oxidation of long-chain fatty acids (LCFAs) is the major source of energy for skeletal muscle. The entry of LCFAs into the mitochondria requires a carnitine-dependent transport system [1] (Figure 1). The so-called carnitine shuttle consists of carnitine palmitoyltransferase I (CPT I), carnitine:acylcarnitine translocase (CACT) and carnitine palmitoyltransferase II (CPT II). CPT I is an integral outer mitochondrial membrane protein and converts acyl-CoAs to acylcarnitines, which are released into the intermembrane space. Acylcarnitines are translocated into the mitochondrial matrix by CACT in exchange with unesterified carnitine. CPT II, located on the inner mitochondrial membrane, catalyzes the reconversion of acylcarnitines to acyl-CoAs, thereby providing substrates for β-oxidation [1,2]
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