Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy.

Abstract

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PPi. STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-D-glycero-D-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from 1H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled 31P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.