[NMR paper] Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments.

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Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments.

Related Articles Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments.

Chembiochem. 2018 Feb 19;:

Authors: Peters T, Weissbach S, Flügge F


Abstract
Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semi-closed", and "closed" conformations upon substrate binding. However, the time scales of corresponding conformational reorientations are unknown. Crystal structures show that Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we have utilized 15N-side chain labeling of Trp residues, and 13C-methyl labeling of Met residues to study substrate induced conformational transitions of GTB. Chemical shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical shift time scale with rather small CSPs in the range of less than ca. 20 Hz. Donor binding matches the intermediate exchange regime yielding an estimate for exchange rate constants around 200 - 300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (