Insights into Allosteric Control of Human Blood Group A and B Glycosyltransferases from Dynamic NMR

Abstract

Human blood group A and B glycosyltransferases (GTA, GTB) are retaining glycosyltransferases, requiring a catalytic mechanism that conserves the anomeric configuration of the hexopyranose moiety of the donor substrate (UDP‐GalNAc, UDP‐Gal). Previous studies have shown that GTA and GTB cycle through structurally distinct states during catalysis. Here, we link binding and release of substrates, substrate‐analogs, and products to transitions between open, semi‐closed, and closed states of the enzymes. Methyl TROSY based titration experiments in combination with zz‐exchange experiments uncover dramatic changes of binding kinetics associated with allosteric interactions between donor‐type and acceptor‐type ligands. Taken together, this highlights how allosteric control of on‐ and off‐rates correlates with conformational changes, driving catalysis to completion.

Structure and Cooperativity in Substrate–Enzyme Interactions: Perspectives on Enzyme Engineering and Inhibitor Design

“…Significant differences in the thermodynamic contributions to the binding of the natural donor compared to that of synthetic analogs have also been confirmed . Recent studies utilizing dynamic NMR performed by Flügge and Peters show, surprisingly, a mutual allosteric cooperativity among the donor and the acceptor substrates, wherein the association and dissociation rates of each regulate those of the other via conformational changes in the enzyme, altogether enhancing the binding affinity of both the substrates to drive the completion of catalysis …”

“…13 Recent studies utilizing dynamic NMR performed by Flugge and Peters show, surprisingly, a mutual allosteric cooperativity among the donor and the acceptor substrates, wherein the association and dissociation rates of each regulate those of the other via conformational changes in the enzyme, altogether enhancing the binding affinity of both the substrates to drive the completion of catalysis. 14 Other studies have reported a negative cooperativity in substrate binding to the enzymes; however, no effect of the negative cooperativity was observed on the enzyme activity. 15 ITC-binding studies and NMR-based structural studies have been extremely useful to study the order of the binding of substrates; substrate-induced conformational change in the enzymes leads to allosteric communication between the substrates that directly affect the enzyme catalysis, 16,17 especially in the oligomeric enzymes, which have multiple catalytic sites on each monomer, and/or modular proteins or enzymes, which have catalytic sites on different domains, and these sites are separated by 10s of Å.…”

Methodological advancements for characterising protein side chains by NMR spectroscopy

“…Employing the sensitive methyl-reporters in macromolecular complexes using methyl-TROSY has become a versatile and generally accessible technique that is now broadly employed to characterise dynamics and allosteric regulation as well as key biological interactions [29][30][31][32][33][34].…”