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 126.96.36.199) 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.