AbstractPeriodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5′‐monophosphate N‐acetylneuraminic acid (CMP‐NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo‐first‐order saturation kinetics, giving a maximum rate of inactivation of 0.6 min–1 and a KI of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with β‐elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme‐oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The β‐elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP This compound also formed a stable reduced morpholino adduct with CMP‐NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its β‐elimination product. Lys19, a residue conserved in CMP‐NeuAc synthetases, was identified as being labeled with the β‐elimination product of oCTP