Reaction of human UMP-CMP kinase with natural and analog substrates.
UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.
Pasti
Claudia
C
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 2185, Institut Pasteur, Paris, France.
Gallois-Montbrun
Sarah
S
Munier-Lehmann
Hélène
H
Veron
Michel
M
Gilles
Anne-Marie
AM
Deville-Bonne
Dominique
D
D001483:Base Sequence; D003001:Cloning, Molecular; D003568:Cytidine Monophosphate / Q000378:metabolism; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014542:Uridine Monophosphate / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014542:Uridine Monophosphate; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D003568:Cytidine Monophosphate