Carnitine O-acetyltransferase - Enzyme Structure

Enzyme Structure

As of late 2007, 7 structures have been solved for this class of enzymes, with PDB accession codes 1NDB, 1NDF, 1NDI, 1NM8, 1S5O, 1T7O, and 1T7Q.

In general, carnitine acetyltransferases have molecular weights of about 70 kDa, and contain approximately 600 residues1. CRAT contains two domains, an N domain and a C domain, and is composed of 20 α helices and 16 β strands. The N domain consists of an eight-stranded β sheet flanked on both sides by eight α helices. A six-stranded mixed β sheet and eleven α helices comprise the enzyme’s C domain.

When compared, the cores of the two domains reflect significantly similar peptide backbone folding. This occurs despite the fact that only 4% of the amino acids that comprise those peptide backbones corresponds to one another.

Active Site: His343 is the catalytic residue in CRAT. It is located at the interface between the enzyme’s C and N domains towards the heart of CRAT. His343 is accessible via two 15-18 Å channels that approach the residue from opposite ends of the CRAT enzyme. These channels are utilized by the substrates of CRAT, one channel for carnitine, and one for CoA. The side chain of His343 is positioned irregularly, with the δ1 ring nitrogen hydrogen bonded to the carbonyl oxygen on the amino acid backbone.

CoA Binding Site: Due to the fact that CRAT binds CoA, rather than acetyl-CoA, it appears that CRAT possesses the ability to hydrolyze acetyl-CoA, before interacting with the lone CoA fragment at the binding site. CoA is bound in a linear conformation with its pantothenic arm binding at the active site. Here, the pantothenic arm’s terminal thiol group and the ε2 nitrogen on the catalytic His343 side chain form a hydrogen bond. The 3’-phosphate on CoA forms interactions with residues Lys419 and Lys423. Also at the binding site, the residues Asp430 and Glu453 form a direct hydrogen bond to one another. If either residue exhibits a mutation, can result in a decrease in CRAT activity.

Carnitine Binding Site: Carnitine binds to CRAT in a partially folded state, with its hydroxyl group and carboxyl group facing opposite directions. The site itself is composed of the C domain β sheet and particular residues from the N domain. Upon binding, a face of carnitine is left exposed to the space outside the enzyme. Like CoA, carnitine forms a hydrogen bond with the ε2 nitrogen on His343. In the case of carnitine, the bond is formed with its 3-hydroxyl group. This CRAT catalysis is stereospecific for carnitine, as the stereoisomer of the 3-hydroxyl group cannot sufficiently interact with the CRAT carnitine binding site. CRAT undergoes minor conformational changes upon binding with carnitine.