Literature summary for 2.7.1.48 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.

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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

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

KM Value [mM]

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

Metals/Ions

Metals/Ions	Comment	Organism
Ca2+	can substitute for Mg2+	Thermus thermophilus
Mg2+	required	Thermus thermophilus
Zn2+	can substitute for Mg2+	Thermus thermophilus

Natural Substrates/ Products (Substrates)

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

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

Purification (Commentary)

Purification (Comment)	Organism
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)

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

Synonyms	Comment	Organism
ttCK	-	Thermus thermophilus
UCK	-	Thermus thermophilus
uridine-cytidine kinase	-	Thermus thermophilus

Temperature Optimum [°C]

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

Temperature Stability [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Thermus thermophilus

Ki Value [mM]

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

General Information

General Information	Comment	Organism
metabolism	uridine-cytidine kinase is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway, overview	Thermus thermophilus
additional information	mechanism underlying nucleoside specificity, overview	Thermus thermophilus
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]

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