William J. Colucci

Carnitine acetyltransferase: A review of its biology, enzymology, and bioorganic chemistry

Abstract The review begins with brief introductory remarks about the significance of carnitine. This is followed by a historical section on its discovery and function, ending with a listing of carnitine-dependent enzymes. Carnitine acetyltransferase then becomes the entire focus of the review. The ubiquity of the protein in tissues and organelles is emphasized in an initial section. A discussion of its enzymology follows, beginning with physical properties and kinetics and ending with substrate and inhibitor specificities. The review concludes with a discussion of proposed molecular mechanisms.

Crystal structures of carnitine and acetylcarnitine zwitterions: A structural hypothesis for mode of action

Abstract The solid-state structures of the zwitterionic forms of both carnitine and acetylcarnitine have been determined by single-crystal X-ray analysis. The crystal structure of acetylcarnitine reveals a different backbone conformation from that of carnitine. The conformational differences observed for carnitine and acetylcarnitine are more a consequence of steric than electrostatic effects. A detailed comparison is made between the zwietterionic structures and previously published hydrochloride salts. The effect of charge distribution on conformation is discussed. The zwitterionic structures do not exhibit enhanced electrostatic attraction between carboxylate and quaternary ammonium portions of the molecules. Finally, a hypothesis is presented for the mode of binding of carnitine (or acetylcarnitine) to the enzyme, carnitine acetyltransferase. Based on this model for binding, a speculative topographic description of the enzymatic mechanism is presented.

Active-site probes of carnitine acyltransferases. Inhibition of carnitine acetyltransferase by hemiacetylcarnitinium, a reaction intermediate analogue.

Hemiacetylcarnitinium (2S,6R:2R,65)-6-carboxymethyl-2-hydroxy-2,4,4- trimethylmorpholinium) chloride is a relatively potent competitive inhibitor (Ki = 0.89 mM) of pigeon breast carnitine acetyltransferase (CAT) and of the crude rat liver CAT (Ki = 4.72 mM) but is neither an inhibitor nor an effective substrate for purified rat liver carnitine palmitoyltransferase (CPT). It does not inhibit state 3 oxygen consumption in isolated hepatic mitochondria using palmitoyl-CoA or palmitoylcarnitine as substrates. This compound is a reaction intermediate analogue of the proposed tetrahedral intermediate for acetyl transfer between acetylcarnitine and CoASH. Because the hemiketal carbon is chiral, a suggestion is made that one of the enantiomers has the same relative configuration as the proposed tetrahedral intermediate.

Preparation of crystalline sodium norcarnitine: An easily handled precursor for the preparation of carnitine analogs and radiolabeled carnitine

A procedure by which crystalline sodium norcarnitine can be prepared in large quantities and high yields has been developed. Carnitine is selectively demethylated by thiophenoxide ion in N,N-dimethylethanolamine. The reactive thiophenoxide ion is generated in situ by addition of thiophenol to this basic reaction solvent. Hence, sodium thiophenoxide, which has been used in similar applications, but is difficult to prepare, can be avoided. Accordingly, reaction of (R,S)-carnitine followed by aqueous azeotropic distillation of byproducts as well as excess starting materials and then by neutralization with sodium hydroxide gave sodium norcarnitine in 89% yield. (R)-Carnitine gave 91% yield of (R)-norcarnitine zwitterion before neutralization. A method for the facile preparation of radiolabeled (R)-carnitine is also described. Thus, methylation of sodium norcarnitine with methyl iodide in methanolic acetone produced carnitine, which precipitated, and sodium iodide, which was soluble.

Hemipalmitoylcarnitinium a strong competitive inhibitor of purified hepatic carnitine palmitoyltransferase

We have synthesized (2S,6R:2R,6S)-6-carboxymethyl-2-hydroxy-2-pentadecyl-4,4-dimethylmorp holinium bromide (hemipalmitoylcarnitinium, HPC) which is a conformationally restricted analog inhibitor of carnitine palmitoyltransferase (CPT; EC 2.3.1.21). rac-HPC inhibits catalytic activity in purified rat liver CPT. In the forward reaction, HPC competes with both (R)-carnitine (Ki(app) = 5.1 +/- 0.7 microM) and palmitoyl-CoA (Ki(app) = 21.5 +/- 4.9 microM). In the reverse reaction, inhibition by HPC is competitive with palmitoyl-(R)-carnitine (Ki(app) = 1.6 +/- 0.6 microM), but inhibition is uncompetitive with CoA. The forward reaction is also competitively inhibited by its product, palmitoyl-(R)-carnitine, Ki(app)s 14.2 +/- 2.1 microM relative to (R)-carnitine and 8.7 +/- 2.6 microM relative to palmitoyl-CoA. rac-HPC is the most potent synthetic reversible inhibitor of purified CPT. HPC fails to inhibit carnitine acetyltransferase (CAT; EC 2.3.1.7). Palmitoylcholine also inhibits CPT in the forward reaction, competing with (R)-carnitine (Ki(app) = 18.6 +/- 4.5 microM) and with palmitoyl CoA (Ki(app) = 10.4 +/- 2.5 microM). Choline is not an effective CPT inhibitor. We have shown [R.D. Gandour et al. (1986) Biochem. Biophys. Res. Commun. 138, 735-741] that hemiacetylcarnitinium inhibits CAT but not CPT. The combined data demonstrate further differences between the carnitine recognition sites in CPT and CAT.