Carnitine O-acetyltransferase
carnitine O-acetyltransferase | |||||||||
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CarnitineAcetylTransferase | |||||||||
Identifiers | |||||||||
EC number | 2.3.1.7 | ||||||||
CAS number | 9029-90-7 | ||||||||
Databases | |||||||||
IntEnz | IntEnz view | ||||||||
BRENDA | BRENDA entry | ||||||||
ExPASy | NiceZyme view | ||||||||
KEGG | KEGG entry | ||||||||
MetaCyc | metabolic pathway | ||||||||
PRIAM | profile | ||||||||
PDB structures | RCSB PDB PDBe PDBsum | ||||||||
Gene Ontology | AmiGO / EGO | ||||||||
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In enzymology, a carnitine O-acetyltransferase (CRAT, or CAT)[1] (EC 2.3.1.7) is an enzyme that catalyzes the chemical reaction
- acetyl-CoA + carnitine CoA + O-acetylcarnitine[2]
Thus, the two substrates of this enzyme are acetyl-CoA and carnitine, whereas its two products are CoA and O-acetylcarnitine.
This enzyme belongs to the family of transferases, to be specific those acyltransferases transferring groups other than aminoacyl groups. The systematic name of this enzyme class is acetyl-CoA:carnitine O-acetyltransferase. Other names in common use include acetyl-CoA-carnitine O-acetyltransferase, acetylcarnitine transferase, carnitine acetyl coenzyme A transferase, carnitine acetylase, carnitine acetyltransferase, carnitine-acetyl-CoA transferase, and CATC. This enzyme participates in alanine and aspartate metabolism.
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.[1]
Active Site: His343 is the catalytic residue in CRAT.[3] 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.[1][4][5]
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.[1] 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.[6][7]
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.[1][8][9]
Enzyme Mechanism
The His343 residue at the active site of CRAT acts as a base that is able to deprotonate the CoA thiol group or the Carnitine 3’-hydroxyl group depending on the direction of the reaction. The structure of CRAT optimizes this reaction by causing direct hydrogen bonding between the His343 and both substrates. The deprotonated group is now free to attack the acetyl group of acetyl-CoA or acetylcarnitine at its carbonyl site. The reaction proceeds directly, without the formation of a His343-acetyl intermediate.
Hydrolysis: It is possible for catalysis to occur with only one of the two substrates. If either acetyl-CoA or acetylcarnitine binds to CRAT, a water molecule may fill the other binding site and act as an acetyl group acceptor.
Substrate-Assisted Catalysis: The literature suggests that the trimethylammonium group on carnitine may be a crucial factor in CRAT catalysis. This group exhibits a positive charge that stabilizes the oxyanion in the reaction’s intermediate. This idea is supported by the fact the positive charge of carnitine is unnecessary for active site binding, but vital for the catalysis to proceed. This has been proven to be the case through the synthesis of a carnitine analog lacking its trimethylammonium group. This compound was able to compete with carnitine in binding to CRAT, but was unable to induce a reaction.[10] The emergence of subtrate-assisted catalysis has opened up new strategies for increasing synthetic substrate specificity.[11]
Biological Function
There is evidence that suggests that CRAT activity is necessary for the cell cycle to proceed from the G1 phase to the S phase.[12]
Disease Relevance
Those with an inherited deficiency in CRAT activity are at risk for developing severe heart and neurological problems.[1]
Reduced CRAT activity can be found in individuals suffering from Alzheimer’s disease.[1]
CRAT and its family of enzymes have great potential as targets for developing therapeutic treatments for Type 2 diabetes and other diseases.[13][14][15]
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Jogl, Gerwald; Tong, Liang (2003). "Crystal Structure of Carnitine Acetyltransferase and Implications for the Catalytic Mechanism and Fatty Acid Transport". Cell 112 (1): 113–22. doi:10.1016/S0092-8674(02)01228-X. PMID 12526798.
- ↑ Bieber, L L (1988). "Carnitine". Annual Review of Biochemistry 57: 261–83. doi:10.1146/annurev.bi.57.070188.001401. PMID 3052273.
- ↑ McGarry, J. Denis; Brown, Nicholas F. (1997). "The Mitochondrial Carnitine Palmitoyltransferase System - from Concept to Molecular Analysis". European Journal of Biochemistry 244 (1): 1–14. doi:10.1111/j.1432-1033.1997.00001.x. PMID 9063439.
- ↑ Jogl, Gerwald; Hsiao, YU-Shan; Tong, Liang (2004). "Structure and Function of Carnitine Acyltransferases". Annals of the New York Academy of Sciences 1033: 17–29. doi:10.1196/annals.1320.002. PMID 15591000.
- ↑ Wu, Donghai; Govindasamy, Lakshmanan; Lian, Wei; Gu, Yunrong; Kukar, Thomas; Agbandje-Mckenna, Mavis; McKenna, Robert (2003). "Structure of Human Carnitine Acetyltransferase. Molecular basis for fatty acyl transfer". Journal of Biological Chemistry 278 (15): 13159–65. doi:10.1074/jbc.M212356200. PMID 12562770.
- ↑ Ramsay, R; Gandour, RD; Van Der Leij, FR (2001). "Molecular enzymology of carnitine transfer and transport". Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1546 (1): 21–43. doi:10.1016/S0167-4838(01)00147-9. PMID 11257506.
- ↑ Hsiao, Y.-S.; Jogl, G; Tong, L (2006). "Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates". Journal of Biological Chemistry 281 (38): 28480–7. doi:10.1074/jbc.M602622200. PMC 2940834. PMID 16870616.
- ↑ Cronin, Ciarán N. (1997). "The Conserved Serine–Threonine–Serine Motif of the Carnitine Acyltransferases is Involved in Carnitine Binding and Transition-State Stabilization: A Site-Directed Mutagenesis Study". Biochemical and Biophysical Research Communications 238 (3): 784–9. doi:10.1006/bbrc.1997.7390. PMID 9325168.
- ↑ Hsiao, Y.-S.; Jogl, G; Tong, L (2004). "Structural and Biochemical Studies of the Substrate Selectivity of Carnitine Acetyltransferase". Journal of Biological Chemistry 279 (30): 31584–9. doi:10.1074/jbc.M403484200. PMID 15155726.
- ↑ Saeed, A; McMillin, JB; Wolkowicz, PE; Brouillette, WJ (1993). "Carnitine Acyltransferase Enzymatic Catalysis Requires a Positive Charge on the Carnitine Cofactor". Archives of Biochemistry and Biophysics 305 (2): 307–12. doi:10.1006/abbi.1993.1427. PMID 8373168.
- ↑ Dall'Acqua, William; Carter, Paul (2008). "Substrate-assisted catalysis: Molecular basis and biological significance". Protein Science 9 (1): 1–9. doi:10.1110/ps.9.1.1. PMC 2144443. PMID 10739241.
- ↑ Brunner, S; Kramar, K; Denhardt, DT; Hofbauer, R (1997). "Cloning and characterization of murine carnitine acetyltransferase: Evidence for a requirement during cell cycle progression". The Biochemical journal 322 (2): 403–10. PMC 1218205. PMID 9065756.
- ↑ Anderson, RC (1998). "Carnitine palmitoyltransferase: A viable target for the treatment of NIDDM?". Current pharmaceutical design 4 (1): 1–16. PMID 10197030.
- ↑ Giannessi, Fabio; Chiodi, Piero; Marzi, Mauro; Minetti, Patrizia; Pessotto, Pompeo; De Angelis, Francesco; Tassoni, Emanuela; Conti, Roberto et al. (2001). "Reversible Carnitine Palmitoyltransferase Inhibitors with Broad Chemical Diversity as Potential Antidiabetic Agents". Journal of Medicinal Chemistry 44 (15): 2383–6. doi:10.1021/jm010889. PMID 11448219.
- ↑ Wagman, A.S.; Nuss, J.M. (2001). "Current Therapies and Emerging Targets for the Treatment of Diabetes". Current Pharmaceutical Design 7 (6): 417–50. doi:10.2174/1381612013397915. PMID 11281851.
Further reading
- Chase, JF; Pearson, DJ; Tubbs, PK (1965). "The Preparation of Crystallin Carnitine Acetyltransferase". Biochimica et Biophysica Acta 96: 162–5. PMID 14285260.
- Friedman, S; Fraenkel, G (1955). "Reversible enzymatic acetylation of carnitine". Archives of Biochemistry and Biophysics 59 (2): 491–501. doi:10.1016/0003-9861(55)90515-4. PMID 13275966.
- Miyazawa, S; Ozasa, H; Furuta, S; Osumi, T; Hashimoto, T (1983). "Purification and properties of carnitine acetyltransferase from rat liver". Journal of biochemistry 93 (2): 439–51. PMID 6404901.