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Literature summary extracted from

  • Tomoike, F.; Nakagawa, N.; Kuramitsu, S.; Masui, R.
    A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine (2011), Biochemistry, 50, 4597-4607.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.48 enzyme H93Y variant ttCK, DNA and amino acid sequence determination and analysis, overexpression in Escherichia coli strain Rosetta(DE3) Thermus thermophilus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.48 purified recombinant enzyme H93Y variant ttCK free or incomplex with CMP, sitting-drop vapor diffusion method, mixing of 500 nl of 25 mg/ml protein, with 1 mM CMP, 1 mM ADP, and 1 mM MgCl2 in case of the enzyme complex, with 500 nl well solution containing 67% 40% v/v isopropanol, 15% w/v PEG 8000, and 0.1 M imidazole, pH 6.5, and 33% distilled water for the free enzyme, and containing 50% w/v PEG 200, and 0.1 M Tris-HCl, pH 8.0, for the enzyme complex, 20°C, X-ray diffraction structure determination and analysis at 2.25 A resolution, molecular replacement Thermus thermophilus

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.48 additional information the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK, due to the naturally occuring polymorphism Y93, the UCK homologue ttCK of Thermus thermophilus HB8 has, unlike other UCKs, a substrate specificity towards only cytidine and shows no inhibition by UTP, uridine does not bind to ttCK as substrate. Replacement of Tyr93 by histidine or glutamine endows ttCK with phosphorylation activity toward uridine Thermus thermophilus

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.1.48 CTP inhibition of wild-type ttCK and mutant Y93H ttCK Thermus thermophilus
2.7.1.48 UTP inhibition of wild-type ttCK Y93, no inhibition of mutant ttCK H93 Thermus thermophilus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.1.48 additional information
-
 additional information Michaelis-Menten kineics Thermus thermophilus
2.7.1.48 0.072
-
 cytidine wild-type ttCK Y93, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.1
-
 cytidine mutant ttCK Y93F, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.2
-
 cytidine mutant ttCK Y59F, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.22
-
 cytidine mutant ttCK Y93H, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.23
-
 cytidine mutant ttCK Y93Q, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.36
-
 uridine mutant ttCK Y93H, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.42
-
 cytidine mutant ttCK Y93L, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 1.6
-
 uridine mutant ttCK Y93Q, pH 8.0, 25°C Thermus thermophilus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.48 Ca2+ can substitute for Mg2+ Thermus thermophilus
2.7.1.48 Mg2+ required Thermus thermophilus
2.7.1.48 Zn2+ can substitute for Mg2+ Thermus thermophilus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.48 ATP + cytidine Thermus thermophilus
-
 ADP + CMP
-
 ?
2.7.1.48 ATP + cytidine Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
 ADP + CMP
-
 ?
2.7.1.48 ATP + uridine Thermus thermophilus
-
 ADP + UMP
-
 ?
2.7.1.48 ATP + uridine Thermus thermophilus substrate of mutant ttCKs Y93H and Y93Q, not of wild-type ttCK Y93 ADP + UMP
-
 ?
2.7.1.48 ATP + uridine Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
 ADP + UMP
-
 ?
2.7.1.48 ATP + uridine Thermus thermophilus HB8 / ATCC 27634 / DSM 579 substrate of mutant ttCKs Y93H and Y93Q, not of wild-type ttCK Y93 ADP + UMP
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.48 Thermus thermophilus
-
-
-
2.7.1.48 Thermus thermophilus Q5SKR5
-
-
2.7.1.48 Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
-
-
2.7.1.48 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 Q5SKR5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.48 recombinant ttCK from Escherichia coli strain Rosetta(DE3) by heat treatment at 60°C for 20 min, hydrophobic interaction chromatography, and gel filtration Thermus thermophilus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.48 ATP + cytidine
-
 Thermus thermophilus ADP + CMP
-
 ?
2.7.1.48 ATP + cytidine
-
 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 ADP + CMP
-
 ?
2.7.1.48 ATP + uridine
-
 Thermus thermophilus ADP + UMP
-
 ?
2.7.1.48 ATP + uridine substrate of mutant ttCKs Y93H and Y93Q, not of wild-type ttCK Y93 Thermus thermophilus ADP + UMP
-
 ?
2.7.1.48 ATP + uridine
-
 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 ADP + UMP
-
 ?
2.7.1.48 ATP + uridine substrate of mutant ttCKs Y93H and Y93Q, not of wild-type ttCK Y93 Thermus thermophilus HB8 / ATCC 27634 / DSM 579 ADP + UMP
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.7.1.48 ttCK
-
 Thermus thermophilus
2.7.1.48 UCK
-
 Thermus thermophilus
2.7.1.48 uridine-cytidine kinase
-
 Thermus thermophilus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.1.48 25
-
 assay at Thermus thermophilus

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.7.1.48 70
-
 enzyme H93Y variant ttCK, stable up to at pH 7.0 Thermus thermophilus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.1.48 0.00118
-
 uridine mutant ttCK Y93Q, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0028
-
 cytidine mutant ttCK Y93Q, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0041
-
 cytidine wild-type ttCK Y93, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0059
-
 cytidine mutant ttCK Y93F, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0064
-
 cytidine mutant ttCK Y59F, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0084
-
 cytidine mutant ttCK Y93H, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0092
-
 uridine mutant ttCK Y93H, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.01
-
 cytidine mutant ttCK Y93L, pH 8.0, 25°C Thermus thermophilus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.1.48 8
-
 assay at Thermus thermophilus

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.48 ATP
-
 Thermus thermophilus

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.7.1.48 0.006
-
 CTP wild-type ttCK (Y93), pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0073
-
 CTP mutant ttCK (Y93H), pH 8.0, 25°C Thermus thermophilus
2.7.1.48 0.0073
-
 UTP mutant ttCK (Y93H), pH 8.0, 25°C Thermus thermophilus

General Information

EC Number General Information Comment Organism
2.7.1.48 metabolism uridine-cytidine kinase is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway, overview Thermus thermophilus
2.7.1.48 additional information mechanism underlying nucleoside specificity, overview Thermus thermophilus
2.7.1.48 additional information mechanism underlying nucleoside specificity, overview. The histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK, due to the naturally occuring polymorphism Y93, the UCK homologue ttCK of Thermus thermophilus HB8 has, unlike other UCKs, a substrate specificity towards only cytidine and shows no inhibition by UTP, uridine does not bind to ttCK as substrate. Replacement of Tyr93 by histidine or glutamine endows ttCK with phosphorylation activity toward uridine Thermus thermophilus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.7.1.48 0.72
-
 uridine mutant ttCK Y93Q, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 12
-
 cytidine mutant ttCK Y93Q, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 24
-
 cytidine mutant ttCK Y93L, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 26
-
 uridine mutant ttCK Y93H, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 32
-
 cytidine mutant ttCK Y59F, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 38
-
 cytidine mutant ttCK Y93H, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 57
-
 cytidine mutant ttCK Y93F, pH 8.0, 25°C Thermus thermophilus
2.7.1.48 57
-
 cytidine wild-type ttCK Y93, pH 8.0, 25°C Thermus thermophilus