Metabolic roles of carnitine expressed through the carnitine acetyltransferase system in Candida pintolopesii

1. 1. The carnitine-responsive mutant yeast, Candida pintolopesii ATCC 26014 and the wild type strain (ATCC 22987) were used to investigate the role of carnitine and the carnitine acetyltransferase system. 2. 2. [ 3H] l-Carnitine, supplied to the cells, was incorporated into acetylcamitine and [ 14C]pantothenate was incorporated into CoA and its derivatives. 3. 3. Both bioautography and quantitative assays indicated that the relative amounts of CoA and acetylCoA were very different in the mutant and wild type cells. 4. 4. The wild type yeast maintained an acetylCoA/CoA ratio of 0.33 ± 0.09 indicating that most of the CoA in the cell is in the free CoA form. Carnitine was not required to establish this ratio nor did its presence lower it further. 5. 5. In contrast, the mutant cells contained a high acetylCoA/CoA ratio (12.8 ± 3.0). 6. 6. In the mutant cells, carnitine lowered the ratio by decreasing the intracellular acetylCoA concentration and releasing free CoA. 7. 7. These data indicated that wild type yeast possess an effective mechanism that is not related to the CAT system for regulating the acetylCoA/CoA ratio. 8. 8. This mechanism appears to be lacking in the mutant. The CAT system decreased the acetylCoA/CoA ratio in the mutant cells but not to the value which is found in the wild type strain. 9. 9. In both stains of Candida pintolopesii, in the presence of carnitine, an acetylcamitine pool can be created whose concentration exceeds that of acetylCoA. 10. 10. The intracellular apparent equilibrium constant ( K app) for carnitine acetyltransferase for wild type Candida pintolopesii ATCC 22987 was 0.73 ± 0.12, close to the established value of 0.6, indicating that the CAT system ran close to equilibrium. 11. 11. The K app for the CAT system of the carnitine-responsive mutant yeast was 7.7 ± 1.7 indicating that this reaction was not at equilibrium.