Characteristics, substrate analysis, and intracellular location of Saccharomyces cerevisiae UMP kinase.
The yeast Saccharomyces cerevisiae SOC8 DNA fragment was isolated as a wildtype dominant suppressor of the cdc8 mutation. The SOC8 has previously been proved to be allelic with URA6, encoding the UMP kinase in yeast cells. The protein has been purified to homogeneity. In this report, we describe the characteristics of the UMP kinase from yeast. The yeast enzyme requires a divalent cation and is active over the entire range of pH from 6 to 9.5. The enzyme can use UMP and dUMP as phosphate acceptors with high activity; to a lesser extent, it can also use IMP, GMP, dGMP, 5-iodo-dUMP, XMP, and dTMP as substrates. ATP and dATP are the best phosphate donors; the enzyme could use GTP, dGTP, dCTP, and dTTP to some degree (30-50%). CTP and UTP were poor phosphate donors for the UMP kinase reaction (10-14%). Like other monophosphate kinases, UMP kinase contains a conserved nucleotide-binding site at its N-terminus following a cysteine residue, and its enzymatic activity is inhibited by sulfhydryl inhibitors such as 5,5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide. Subcellular localization studies indicate that the UMP kinase locates primarily in the cytoplasm (approximately 80%) and also in the nucleus (approximately 20%), but not in the mitochondria. These results suggest that it may exert its function in the nucleus, such as in RNA synthesis, as well as in the cytoplasm, but not in the mitochondria. The presence of UMP kinase in the nucleus might facilitate its suppression of cdc8 mutant cells, which are defective in nuclear DNA synthesis.
Jong
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A
Department of Pediatrics and Microbiology, University of Southern California School of Medicine, Los Angeles 90033.
Yeh
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Y
Ma
J J
JJ
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D002413:Cations, Divalent; D010710:Phosphates; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase