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Literature summary for 2.7.4.22 extracted from

  • Egeblad-Welin, L.; Welin, M.; Wang, L.; Eriksson, S.
    Structural and functional investigations of Ureaplasma parvum UMP kinase--a potential antibacterial drug target (2007), FEBS J., 274, 6403-6414.
    View publication on PubMed

Activating Compound

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

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(Commentary)

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 (Commentary)

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

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

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 [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

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/ Products (Substrates)

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

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 (Commentary)

Purification (Comment) Organism
metal affinity column using the gravity flow procedure, purity determined by SDS-PAGE Ureaplasma parvum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + UMP two-substrate kinetics with UMP and ATP Ureaplasma parvum ADP + UDP
-
r

Subunits

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

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.8
-
-
Ureaplasma parvum
6.8
-
purified recombinant enzyme Ureaplasma parvum

Cofactor

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 [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 IC50 Value Maximum Comment Organism Inhibitor Structure
1
-
phosphate ion is found in the active site Ureaplasma parvum phosphate