Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | GTP has no detectable effect on UpUMPK activity unlike UMPKs from other bacteria | Ureaplasma parvum | |
additional information | no activation of wild-type enzyme and F133 mutants by GTP | Ureaplasma parvum |
Application | Comment | Organism |
---|---|---|
drug development | potential drug target for development of antimicrobial agents, diseases such as urethritis and prostatitis | Ureaplasma parvum |
drug development | the enzyme is a potential antibacterial drug target | Ureaplasma parvum |
Cloned (Comment) | Organism |
---|---|
gene pyrH, DNA and amino acid sequence determination, analysis, and comparisons | Ureaplasma parvum |
the mutants UpUMPKF133N and UpUMPK-F133A are constructed by site-directed mutagenesis using the plasmid pET-14b containing cDNA for UMPK, expression in Escherichia coli BL21 (DE3), overexpression by induction with isopropyl-beta-D-thiogalactoside | Ureaplasma parvum |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme, X-ray diffraction structure determination and analysis at 2.5 A resolution | Ureaplasma parvum |
UMPK with N-terminal His-tag, in complex with a phosphate ion, co-crystallized with UMP, and UTP, cross-talk region between two subunits of UpUMPK is identified, vapor diffusion, hanging drop, 0.2 m ammonium fluoride and 20% (w/v) poly(ethylene glycol) 3350, 15°C, enzyme concentration is 1.8 mg/ml, 5 mM GTP, resolution of 2.5 A, space group P 1 21 1, cell dimensions a =79.8, b = 96.6, c = 96.3 A, beta = 105.8 | Ureaplasma parvum |
Protein Variants | Comment | Organism |
---|---|---|
F133A | site-directed mutagenesis, activity is only 20% of the wild-type, still no activation by GTP, with variable UMP concentration and fixed ATP concentration exhibits negative cooperativity with UMP, Hill coefficient 0.85 | Ureaplasma parvum |
F133A | site-directed mutagenesis, the mutant enzyme is not activated by GTP and exhibits negative cooperativity with UMP | Ureaplasma parvum |
F133N | site-directed mutagenesis, activity is only 50% of the wild-type, still no activation by GTP, demonstrating that F133N is involved in subunit interactions but apparently not in GTP activation, with variable UMP concentration and fixed ATP concentration exhibits negative cooperativity with UMP, Hill coefficient 0.65, marked decrease in activity | Ureaplasma parvum |
F133N | site-directed mutagenesis, the mutant enzyme is not activated by GTP and exhibits negative cooperativity with UMP | Ureaplasma parvum |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
phosphate | phosphate-inhibited UpUMPK activity with an IC50 value of 1 mM, the molar content of phosphate in the soluble UMPK is 4% | Ureaplasma parvum | |
UTP | an end-product inhibitor competitive against UMP and noncompetitive towards ATP; UTP is a competitive inhibitor against UMP and a noncompetitive inhibitor towards ATP, the positive cooperativity behavior with ATP is altered by the presence of UTP | Ureaplasma parvum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | with ATP as the variable substrate, the kinetic curves show a detectable deviation from Michaelis-Menten kinetics, especially at low substrate concentrations, the best fit is therefore to the Hill equation, giving an n value of 1.54 +/- 0.10, demonstrating positive cooperativity with ATP, coupled spectrophotometric assay | Ureaplasma parvum | |
additional information | - |
additional information | MPK exhibited Michaelis-Menten kinetics | Ureaplasma parvum | |
0.214 | - |
UMP | pH 6.8 | Ureaplasma parvum | |
0.214 | - |
UMP | recombinant UpUMPK | Ureaplasma parvum |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | a stoichiometry between Mg2+ and ATP of 2:1 gave 1.6fold higher catalytic rates, compared to a 1:1 stoichiometry | Ureaplasma parvum | |
Mg2+ | a stoichiometry between Mg2+ and ATP of 2:1 gives 1.6fold higher catalytic rates compared to a 1:1 stoichiometry | Ureaplasma parvum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + UMP | Ureaplasma parvum | two-substrate kinetics with UMP and ATP | ADP + UDP | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Ureaplasma parvum | Q9PPX6 | plasmid pET-14b containing cDNA for UMPK, expression in Escherichia coli BL21 (DE3), overexpression by induction with isopropyl-b-d-thiogalactoside | - |
Ureaplasma parvum | Q9PPX6 | gene pyrH | - |
Purification (Comment) | Organism |
---|---|
metal affinity column using the gravity flow procedure, purity determined by SDS-PAGE | Ureaplasma parvum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + UMP | two-substrate kinetics with UMP and ATP | Ureaplasma parvum | ADP + UDP | - |
r |
Subunits | Comment | Organism |
---|---|---|
hexamer | trimer of dimers, crystallization, the forces that hold the hexamer together are (a) hydrophobic interactions between the dimeric couples A + B, C + D and E + F (b) a few hydrogen bonds, and a hydrophobic interaction between A + C, B + E and D + F, and (c) electrostatic forces in the central channel of the hexamer between B, C and F, and A, D and E | Ureaplasma parvum |
More | three-dimensional structure, residue F133 is involved in subunit interaction, the monomer subunit consists of an alpha? beta-fold with a nine-stranded twisted beta-sheet surrounded by eight alpha-helices and one 310 helix, overview | Ureaplasma parvum |
Synonyms | Comment | Organism |
---|---|---|
More | bacterial and archaeal UMPKs all belong to the amino acid kinase family | Ureaplasma parvum |
UMP kinase | - |
Ureaplasma parvum |
UMPK | - |
Ureaplasma parvum |
uridine monophosphate kinase | - |
Ureaplasma parvum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6.8 | - |
- |
Ureaplasma parvum |
6.8 | - |
purified recombinant enzyme | Ureaplasma parvum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | a stoichiometry between Mg2+ and ATP of 2:1 gives 1.6fold higher catalytic rates compared to a 1:1 stoichiometry | Ureaplasma parvum |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | inhibition knetics | Ureaplasma parvum | |
0.7 | - |
UTP | competitive inhibitor towards UMP | Ureaplasma parvum | |
0.7 | - |
UTP | versus UMP, pH 6.8 | Ureaplasma parvum | |
1.2 | - |
UTP | noncompetitive inhibitor towards ATP | Ureaplasma parvum | |
1.2 | - |
UTP | versus ATP, pH 6.8 | Ureaplasma parvum |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
1 | - |
phosphate ion is found in the active site | Ureaplasma parvum | phosphate |