Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates.
Pyrimidine nucleoside monophosphate kinase [UMP/CMP kinase (UMP/CMPK);EC 2.7.4.14] plays a crucial role in the formation of UDP, CDP, and dCDP, which are required for cellular nucleic acid synthesis. Several cytidine and deoxycytidine analogues are important anticancer and antiviral drugs. These drugs require stepwise phosphorylation to their triphosphate forms to exert their therapeutic effects. The role of UMP/CMPK for the phosphorylation of nucleoside analogues has been indicated. Thus, we cloned the human UMP/CMPK gene, expressed it in Escherichia coli, and purified it to homogeneity. Its kinetic properties were determined. UMP and CMP proved to be far better substrates than dCMP. UMP/CMPK used all of the nucleoside triphosphates as phosphate donors, with ATP and dATP being the best donors and CTP being the poorest. Furthermore, UMP/CMPK was able to phosphorylate all of the deoxycytidine analogue monophosphates that we tested. The relative efficiency was as follows: arabinofuranosyl-CMP > dCMP > beta-L-2',3'-dideoxy-3'-thia-CMP > Gemcitabine monophosphate > beta-D-2',3'-dideoxy-CMP; beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluoro-CMP; beta-L-2',3'-dideoxy-5-fluoro-3'-thia-CMP > beta-L-2',3'-dideoxy-CMP > beta-L-dioxolane-CMP. By comparing the relative V(max)/K(m) values of D- and L-form dideoxy-CMP, we showed that this kinase lacked stereoselectivity. Reducing agents, such as DTT, 2-mercaptoethanol, and thioredoxin, were able to activate this enzyme, suggesting that its activity may be regulated by redox potential in vivo. UMP/CMPK localized predominantly to the cytoplasm. In addition, 196-amino acid UMP/CMPK was the actual form of UMP/CMPK, rather than the 228-amino acid form as suggested before.
Liou
Jieh-Yuan
JY
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Dutschman
Ginger E
GE
Lam
Wing
W
Jiang
Zaoli
Z
Cheng
Yung-Chi
YC
D000918:Antibody Specificity; D003001:Cloning, Molecular; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011743:Pyrimidines / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013347:Subcellular Fractions / Q000201:enzymology
D011743:Pyrimidines; D011994:Recombinant Proteins; D003841:Deoxycytidine; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D003568:Cytidine Monophosphate