Characterization of specific donor binding to alpha1,4-N-acetylhexosaminyltransferase EXTL2 using isothermal titration calorimetry.
Glycosyltransferases encompass one of the largest families of enzymes found in nature. Their principle function is to catalyze the transfer of activated donor-sugar molecules to various acceptor substrates. The molecular basis that governs this specific transfer reaction, such as how a given transferase determines donor-sugar specificity, remains to be elucidated. Human alpha1,4-N-acetylhexosaminyltransferase (EXTL2) transfers N-acetylglucosamine and N-acetylgalactosamine but does not transfer glucose or galactose. Isothermal titration calorimetry (ITC) is a powerful technique used to characterize various binding reactions, including both protein-ligand and protein-protein interactions. ITC provides the binding stoichiometry, affinity, and the thermodynamic parameters free energy (DeltaG), enthalpy (DeltaH), and entropy (DeltaS) of these binding interactions. This chapter describes our ITC study demonstrating the two-step mechanism that regulates the specific binding of N-acetylhexosamines to EXTL2.
Sobhany
Mack
M
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, NC, USA.
Negishi
Masahiko
M
D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics
D008565:Membrane Proteins; C110834:EXTL2 protein, human; D017351:N-Acetylglucosaminyltransferases; D000117:Acetylglucosamine