Covalent modification of Lys19 in the CTP binding site of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase

Abstract

Periodate 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 K_I 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.