Literature summary for 2.7.3.2 extracted from

Cox, J.M.; Davis, C.A.; Chan, C.; Jourden, M.J.; Jorjorian, A.D.; Brym, M.J.; Snider, M.J.; Borders, C.L., Jr.; Edmiston, P.L.
Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability (2003), Biochemistry, 42, 1863-1871.

Search for more literature for 2.7.3.2

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression of soluble wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), triple mutant enzyme R147A/R151A/D209A is expressed as insoluble, aggregated protein	Oryctolagus cuniculus

Protein Variants

Protein Variants	Comment	Organism
D209A	site-directed mutagenesis, mutant enzyme appears as a mixture of monomeric and dimeric forms, the monomer shows higher thermolability and sensitivity aginst unfolding by 1-anilinonaphthalene-8-sulfonate due to a higher surface area	Oryctolagus cuniculus
R147A	site-directed mutagenesis, mutant enzyme is a monomer showing higher thermolability and sensitivity aginst unfolding by 1-anilinonaphthalene-8-sulfonate due to a higher surface area, reduced activity and 89% reduced kcat compared to the wild-type enzyme, the mutant enzyme does not follow a random-order rapid-equilibrium mechanism like the wild-type enzyme, but to an ordered mechanism with creatine binding first	Oryctolagus cuniculus
R147A/R151A	site-directed mutagenesis, double mutant enzyme is a monomer showing higher thermolability and sensitivity aginst unfolding by 1-anilinonaphthalene-8-sulfonate due to a higher surface area, 10fold reduced substrate binding and 40% reduced kcat compared to the wild-type enzyme, the mutant enzyme follows a random-order rapid-equilibrium mechanism like the wild-type enzyme	Oryctolagus cuniculus
R147A/R151A/D209A	site-directed mutagenesis, the triple mutant enzyme is expressed as insoluble, aggregated protein	Oryctolagus cuniculus
R151A	site-directed mutagenesis, mutant enzyme is a dimer, reduced activity compared to the wild-type enzyme	Oryctolagus cuniculus

Inhibitors

Inhibitors	Comment	Organism	Structure
1-anilinonaphthalene-8-sulfonate	unfolding agent	Oryctolagus cuniculus
guanidinium hydrochloride	inactivation mechanism of wild-type and mutant enzymes, overview	Oryctolagus cuniculus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	kinetics	Oryctolagus cuniculus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
cytosol	-	Oryctolagus cuniculus	5829	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Oryctolagus cuniculus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
42000	-	recombinant mutant R147A, analytical ultracentrifugation	Oryctolagus cuniculus
45000	-	recombinant mutant R147A/R151A, gel filtration and analytical ultracentrifugation	Oryctolagus cuniculus
46000	-	recombinant mutant R147A, gel filtration	Oryctolagus cuniculus
47000	-	1 * 47000, about, recombinant mutant enzymes D209A, R147A, and R147A/R151A, SDS-PAGE	Oryctolagus cuniculus
47000	-	2 * 47000, about, recombinant wild-type enzyme and recombinant mutant enzymes D209A and R151A, SDS-PAGE	Oryctolagus cuniculus
67000	-	recombinant mutant R151A, analytical ultracentrifugation	Oryctolagus cuniculus
71000	-	recombinant mutant D209A, analytical ultracentrifugation	Oryctolagus cuniculus
74000	-	recombinant mutant D209A, gel filtration	Oryctolagus cuniculus
81000	-	recombinant mutant R151A, gel filtration	Oryctolagus cuniculus
85000	-	recombinant wild-type enzyme, gel filtration	Oryctolagus cuniculus
86000	-	recombinant wild-type enzyme, analytical ultracentrifugation	Oryctolagus cuniculus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + creatine	Oryctolagus cuniculus	-	ADP + phosphocreatine	-	r

Organism

Organism	UniProt	Comment	Textmining
Oryctolagus cuniculus	P00563	cytosolic isozyme	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)	Oryctolagus cuniculus

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + creatine = ADP + phosphocreatine	random-order rapid-equilibrium mechanism	Oryctolagus cuniculus	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
skeletal muscle	-	Oryctolagus cuniculus	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + creatine	-	Oryctolagus cuniculus	ADP + phosphocreatine	-	r

Subunits

Subunits	Comment	Organism
dimer	2 * 47000, about, recombinant wild-type enzyme and recombinant mutant enzymes D209A and R151A, SDS-PAGE	Oryctolagus cuniculus
monomer	1 * 47000, about, recombinant mutant enzymes D209A, R147A, and R147A/R151A, SDS-PAGE	Oryctolagus cuniculus
More	dimerization is no prerequisite for activity but is required for stability and quarternary structure, subunit interface structure and interacting side chains of Glu17, Tyr19, sp53, Gln57, Asp61, Asp209, Arg147, and Arg151, overview	Oryctolagus cuniculus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Oryctolagus cuniculus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
37	-	inactivation of recombinant mutant R147A/R151A	Oryctolagus cuniculus
40	43	inactivation of recombinant mutant R151A	Oryctolagus cuniculus
41	-	inactivation of recombinant mutant R147A	Oryctolagus cuniculus
53	55	inactivation of recombinant wild-type enzyme	Oryctolagus cuniculus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
9	-	assay at	Oryctolagus cuniculus

Cofactor

Cofactor	Comment	Organism	Structure
ADP	-	Oryctolagus cuniculus
ATP	-	Oryctolagus cuniculus

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Release 2023.1 (January 2023)