Uridine kinase: altered enzyme with decreased affinities for uridine and CTP.
Uridine kinase is the rate-limiting enzyme in the salvage pathway for uridine or cytidine of mammalian cells. Alignment of the uridine kinase sequence with other nucleoside and nucleotide kinases supports a common ancestor for all of these. Three polypeptide segments for the ATP site and three polypeptide segments for the acceptor nucleoside site have been identified. We report here the characterization of an altered form of the enzyme with a single amino acid change, Q146R, within or near the uridine-binding site. This single amino acid change leads to a 160-fold increase in Km for uridine (Km = 6.5 mM) and a decrease in kcat by more than 99%. This variant has normal affinity for ATP (Km = 130 microM), but shows substrate inhibition at ATP concentrations >3 mM. Mouse uridine kinase is normally an active tetramer that will dissociate to inactive monomers in response to CTP. In contrast, the altered protein is monomeric, but will associate to dimers and then to tetramers with increasing ATP. The Q146R enzyme has a 100-fold loss in affinity for the allosteric inhibitor CTP; this supports a model for CTP inhibition being caused by CTP binding backward at the catalytic site, as a bisubstrate analog.
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Department of Biochemistry & Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina, 27599-7260, USA.
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D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003570:Cytidine Triphosphate / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D014529:Uridine / Q000378:metabolism*; D014541:Uridine Kinase / Q000235:genetics* / Q000378:metabolism
D003570:Cytidine Triphosphate; D014541:Uridine Kinase; D014529:Uridine