This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Liou, J.-Y. Articles by Cheng, Y.-C. Search for Related Content PubMed PubMed Citation Articles by Liou, J.-Y. Articles by Cheng, Y.-C.

Characterization of Human UMP/CMP Kinase and Its Phosphorylation of D- and L-Form Deoxycytidine Analogue Monophosphates¹

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520

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 > ß-L-2',3'-dideoxy-3'-thia-CMP > Gemcitabine monophosphate > ß-D-2',3'-dideoxy-CMP; ß-L-2',3'-dideoxy-2',3'-didehydro-5-fluoro-CMP; ß-L-2',3'-dideoxy-5-fluoro-3'-thia-CMP > ß-L-2',3'-dideoxy-CMP > ß-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.

This article has been cited by other articles:

				X. Wang, H. Tanaka, M. Baba, and Y.-c. Cheng Retention of Metabolites of 2',3'-Didehydro-3'-Deoxy-4'-Ethynylthymidine, a Novel Anti-Human Immunodeficiency Virus Type 1 Thymidine Analog, in Cells Antimicrob. Agents Chemother., August 1, 2009; 53(8): 3317 - 3324. [Abstract] [Full Text] [PDF]

				R. Humeniuk, L. G. Menon, P. J. Mishra, R. Gorlick, R. Sowers, W. Rode, G. Pizzorno, Y.-C. Cheng, N. Kemeny, J. R. Bertino, et al. Decreased levels of UMP kinase as a mechanism of fluoropyrimidine resistance Mol. Cancer Ther., May 1, 2009; 8(5): 1037 - 1044. [Abstract] [Full Text] [PDF]

				C. Thum, C. Z. Schneider, M. S. Palma, D. S. Santos, and L. A. Basso The Rv1712 Locus from Mycobacterium tuberculosis H37Rv Codes for a Functional CMP Kinase That Preferentially Phosphorylates dCMP J. Bacteriol., April 15, 2009; 191(8): 2884 - 2887. [Abstract] [Full Text] [PDF]

				W. Lam, S. Bussom, and Y.-C. Cheng Effect of hypoxia on the expression of phosphoglycerate kinase and antitumor activity of troxacitabine and gemcitabine in non-small cell lung carcinoma Mol. Cancer Ther., February 1, 2009; 8(2): 415 - 423. [Abstract] [Full Text] [PDF]

				N. A. Kocabas, P. Aksoy, L. L. Pelleymounter, I. Moon, J.-S. Ryu, J. A. Gilbert, O. E. Salavaggione, B. W. Eckloff, E. D. Wieben, V. Yee, et al. Gemcitabine Pharmacogenomics: Deoxycytidine Kinase and Cytidylate Kinase Gene Resequencing and Functional Genomics Drug Metab. Dispos., September 1, 2008; 36(9): 1951 - 1959. [Abstract] [Full Text] [PDF]

				Y. Xu, M. Johansson, and A. Karlsson Human UMP-CMP Kinase 2, a Novel Nucleoside Monophosphate Kinase Localized in Mitochondria J. Biol. Chem., January 18, 2008; 283(3): 1563 - 1571. [Abstract] [Full Text] [PDF]

				W. Lam, C.-H. Leung, S. Bussom, and Y.-C. Cheng The Impact of Hypoxic Treatment on the Expression of Phosphoglycerate Kinase and the Cytotoxicity of Troxacitabine and Gemcitabine Mol. Pharmacol., September 1, 2007; 72(3): 536 - 544. [Abstract] [Full Text] [PDF]

				J. A.C. Alexandre, B. Roy, D. Topalis, S. Pochet, C. Perigaud, and D. Deville-Bonne Enantioselectivity of human AMP, dTMP and UMP-CMP kinases Nucleic Acids Res., July 10, 2007; (2007) gkm479v1. [Abstract] [Full Text] [PDF]

				C.-H. Hsu, R. Hu, G. E. Dutschman, G. Yang, P. Krishnan, H. Tanaka, M. Baba, and Y.-C. Cheng Comparison of the Phosphorylation of 4'-Ethynyl 2',3'-Dihydro-3'-Deoxythymidine with That of Other Anti-Human Immunodeficiency Virus Thymidine Analogs Antimicrob. Agents Chemother., May 1, 2007; 51(5): 1687 - 1693. [Abstract] [Full Text] [PDF]

				E. Murakami, H. Bao, M. Ramesh, T. R. McBrayer, T. Whitaker, H. M. Micolochick Steuer, R. F. Schinazi, L. J. Stuyver, A. Obikhod, M. J. Otto, et al. Mechanism of Activation of {beta}-D-2'-Deoxy-2'-Fluoro-2'-C-Methylcytidine and Inhibition of Hepatitis C Virus NS5B RNA Polymerase Antimicrob. Agents Chemother., February 1, 2007; 51(2): 503 - 509. [Abstract] [Full Text] [PDF]

				Z. Zhou, J. H. Rodman, P. M. Flynn, B. L. Robbins, C. K. Wilcox, and D. Z. D'Argenio Model for intracellular Lamivudine metabolism in peripheral blood mononuclear cells ex vivo and in human immunodeficiency virus type 1-infected adolescents. Antimicrob. Agents Chemother., August 1, 2006; 50(8): 2686 - 2694. [Abstract] [Full Text] [PDF]

				L. P. Jordheim, O. Guittet, M. Lepoivre, C. M. Galmarini, and C. Dumontet Increased expression of the large subunit of ribonucleotide reductase is involved in resistance to gemcitabine in human mammary adenocarcinoma cells Mol. Cancer Ther., August 1, 2005; 4(8): 1268 - 1276. [Abstract] [Full Text] [PDF]

				R. Hu, L. Li, B. Degreve, G. E. Dutschman, W. Lam, and Y.-C. Cheng Behavior of Thymidylate Kinase toward Monophosphate Metabolites and Its Role in the Metabolism of 1-(2'-Deoxy-2'-Fluoro-{beta}-L-Arabinofuranosyl)-5-Methyluracil (Clevudine) and 2',3'-Didehydro-2',3'-Dideoxythymidine in Cells Antimicrob. Agents Chemother., May 1, 2005; 49(5): 2044 - 2049. [Abstract] [Full Text] [PDF]

				C.-H. Hsu, J.-Y. Liou, G. E. Dutschman, and Y.-C. Cheng Phosphorylation of Cytidine, Deoxycytidine, and Their Analog Monophosphates by Human UMP/CMP Kinase Is Differentially Regulated by ATP and Magnesium Mol. Pharmacol., March 1, 2005; 67(3): 806 - 814. [Abstract] [Full Text] [PDF]

				L. P. Jordheim, E. Cros, M.-H. Gouy, C. M. Galmarini, S. Peyrottes, J. Mackey, C. Perigaud, and C. Dumontet Characterization of a Gemcitabine-Resistant Murine Leukemic Cell Line: Reversion of In vitro Resistance by a Mononucleotide Prodrug Clin. Cancer Res., August 15, 2004; 10(16): 5614 - 5621. [Abstract] [Full Text] [PDF]

				D. Segura-Pena, N. Sekulic, S. Ort, M. Konrad, and A. Lavie Substrate-induced Conformational Changes in Human UMP/CMP Kinase J. Biol. Chem., August 6, 2004; 279(32): 33882 - 33889. [Abstract] [Full Text] [PDF]

				T. E. Kim, S.-Y. Park, C.-H. Hsu, G. E. Dutschman, and Y.-C. Cheng Synergistic Antitumor Activity of Troxacitabine and Camptothecin in Selected Human Cancer Cell Lines Mol. Pharmacol., August 1, 2004; 66(2): 285 - 292. [Abstract] [Full Text] [PDF]

				H. Sakamoto, S. Landais, C. Evrin, C. Laurent-Winter, O. Barzu, and R. A. Kelln Structure-function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria Microbiology, July 1, 2004; 150(7): 2153 - 2159. [Abstract] [Full Text] [PDF]

				P. Krishnan, E. A. Gullen, W. Lam, G. E. Dutschman, S. P. Grill, and Y.-c. Cheng Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents J. Biol. Chem., September 19, 2003; 278(38): 36726 - 36732. [Abstract] [Full Text] [PDF]

				J.-Y. Liou, P. Krishnan, C.-C. Hsieh, G. E. Dutschman, and Y.-c. Cheng Assessment of the Effect of Phosphorylated Metabolites of Anti-Human Immunodeficiency Virus and Anti-Hepatitis B Virus Pyrimidine Analogs on the Behavior of Human Deoxycytidylate Deaminase Mol. Pharmacol., January 1, 2003; 63(1): 105 - 110. [Abstract] [Full Text] [PDF]