4eyw Citations

Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein.

OpenAccess logo FEBS Open Bio 3 204-11 (2013)
Related entries: 4ep9, 4eph

Cited: 8 times
EuropePMC logo PMID: 23772395


Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by β-octylglucoside (β-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of K D = 2-20 μM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery.

Reviews citing this publication (2)

  1. Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015. Falconer RJ. J. Mol. Recognit. 29 504-515 (2016)
  2. Mitochondrial biology, targets, and drug delivery. Milane L, Trivedi M, Singh A, Talekar M, Amiji M. J Control Release 207 40-58 (2015)

Articles citing this publication (6)

  1. Characterization of membrane protein interactions by isothermal titration calorimetry. Situ AJ, Schmidt T, Mazumder P, Ulmer TS. J. Mol. Biol. 426 3670-3680 (2014)
  2. Determination of thermodynamic potentials and the aggregation number for micelles with the mass-action model by isothermal titration calorimetry: A case study on bile salts. Olesen NE, Westh P, Holm R. J Colloid Interface Sci 453 79-89 (2015)
  3. Isothermal titration calorimetry of ion-coupled membrane transporters. Boudker O, Oh S. Methods 76 171-182 (2015)
  4. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids. Touchette MH, Bommineni GR, Delle Bovi RJ, Gadbery JE, Nicora CD, Shukla AK, Kyle JE, Metz TO, Martin DW, Sampson NS, Miller WT, Tonge PJ, Seeliger JC. Biochemistry 54 5457-5468 (2015)
  5. Simple ITC method for activity and inhibition studies on human salivary α-amylase. Lehoczki G, Szabó K, Takács I, Kandra L, Gyémánt G. J Enzyme Inhib Med Chem 31 1648-1653 (2016)
  6. Tuning the photoexcitation response of cyanobacterial Photosystem I via reconstitution into Proteoliposomes. Niroomand H, Mukherjee D, Khomami B. Sci Rep 7 2492 (2017)