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 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 beta-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 beta-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 beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP.
Tullius
M V
MV
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Vann
W F
WF
Gibson
B W
BW
D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry; D000216:N-Acylneuraminate Cytidylyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D013379:Substrate Specificity
D003570:Cytidine Triphosphate; D000216:N-Acylneuraminate Cytidylyltransferase; D008239:Lysine