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

Periodate oxidized CTP (oCTP) was used to investigate the importance of lys ine residues in the CTP binding site of the cytidine 5'-monophosphate N-ace tylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus du creyi. The reaction of oCTP with the enzyme follows pseudo-first-order satu ration 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 enzym e provided data that was consistent with beta-elimination of triphosphate a fter the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conj ugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH 3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentra tions 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 subs trate CTP was capable of protecting the enzyme to a large degree from inact ivation by oCTP and its beta-elimination product. Lys19, a residue conserve d in CMP-NeuAc synthetases, was identified as being labeled with the beta-e limination product of oCTP.