title: Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria.
abstract: In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.
authors.affiliation: UnitédeGénétique des Génomes Bactériens, Institut Pasteur, 75724 Paris Cedex, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Evrin
authors.forename: Monica
authors.initials: M
authors.lastname: Straut
authors.forename: Neli
authors.initials: N
authors.lastname: Slavova-Azmanova
authors.forename: Nadia
authors.initials: N
authors.lastname: Bucurenci
authors.forename: Adrian
authors.initials: A
authors.lastname: Onu
authors.forename: Liliane
authors.initials: L
authors.lastname: Assairi
authors.forename: Mihaela
authors.initials: M
authors.lastname: Ionescu
authors.forename: Nicolae
authors.initials: N
authors.lastname: Palibroda
authors.forename: Octavian
authors.initials: O
authors.lastname: Bârzu
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Gilles
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; D014544:Uridine Triphosphate