Two-step mechanism that determines the donor binding specificity of human UDP-N-acetylhexosaminyltransferase.
In its x-ray crystal structures, alpha-1,4-N-acteylhexosaminyltransferase (exostosin-like protein 2 (EXTL2)) forms no direct interaction with the N-acetyl group of the UDP-N-acetylhexosamine. Mutation of the residues that interact with the hydroxyl groups of the donor not only failed to abrogate donor binding but in fact increased binding affinity. Isothermal titration calorimetry is now used to examine the binding nature of various UDP-sugars in H2O and D2O solutions. UDP-N-acetylhexosamines bind to EXTL2 with a high affinity in both solutions, resulting in a relatively large increase of entropy, whereas the weak binding of UDP-galactose and -glucose, which occurred only in D2O solution, only slightly increased entropy. Thus, specific donor binding appears to undergo two distinct steps, beginning with the N-acetyl group expelling water from the donor. enzyme complex into the bulk solvent followed by positioning of the donor into the binding site for the subsequent interactions with the enzyme.
Sobhany
Mack
M
Pharmacogenetic Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Dong
Jian
J
Negishi
Masahiko
M
D001483:Base Sequence; D002151:Calorimetry; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
D017931:DNA Primers; D008565:Membrane Proteins; C110834:EXTL2 protein, human; D017351:N-Acetylglucosaminyltransferases