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Literature summary for 2.7.3.2 extracted from

  • Novak, W.R.P.; Wang, P.F.; McLeish, M.J.; Kenyon, G.L.; Babbitt, P.C.
    Isoleucine 69 and valine 325 form a specificity pocket in human muscle creatine kinase (2004), Biochemistry, 43, 13766-13774.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Homo sapiens

Protein Variants

Protein Variants Comment Organism
I69A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
I69L site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
I69V site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme Homo sapiens
V325A site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme, mutant shows a slight preference for cyclocreatine, i.e. 1-carboxymethy-2-iminoimidazolidine, as substrate Homo sapiens
V325E site-directed mutagenesis, altered substrate specificity compared to the wild-type enzyme, mutant shows a more than 100fold higher preference for N-ethylglycocyamine as substrate compared to creatine, highly reduced activity with other substrates compared to the wild-type enzyme Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics, recombinant wild-type and mutant enzymes Homo sapiens
0.22
-
ATP pH 9.0, 30°C, recombinant wild-type enzyme, with substrate creatine Homo sapiens
0.22
-
ATP ATP in form of MgATP2- Homo sapiens
0.29
-
ATP pH 9.0, 30°C, recombinant wild-type enzyme, with substrate cyclocreatine Homo sapiens
0.29
-
ATP ATP in form of MgATP2- Homo sapiens
0.62
-
ATP pH 9.0, 30°C, recombinant wild-type enzyme, with substrate N-ethylglycocyamine Homo sapiens
0.62
-
ATP ATP in form of MgATP2- Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
Homo sapiens

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Homo sapiens P12277
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
ATP + creatine = ADP + phosphocreatine catalytic mechanism and substrate binding involving 2 flexible loops and a specificity pocket formed by Ile69 and Val325 determining substrate specificity, active site structure Homo sapiens

Source Tissue

Source Tissue Comment Organism Textmining
skeletal muscle
-
Homo sapiens
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
substrate specificities of recombinant wild-type and mutant enzymes Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + creatine
-
Homo sapiens ADP + phosphocreatine
-
r
ATP + cyclocreatine i.e. 1-carboxymethyl-2-iminoimidazolidine Homo sapiens ADP + phospho-cyclocreatine
-
?
ATP + glycocyamine very low activity Homo sapiens ADP + glycocyamine phosphate
-
?
ATP + N-ethylglycocyamine
-
Homo sapiens ADP + N-ethylglycocyamine phosphate
-
?

Synonyms

Synonyms Comment Organism
More the enzyme is a member of the phosphagen kinase superfamily of enzymes Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.55
-
N-ethylglycocyamine pH 9.0, 30°C, recombinant mutant I69L Homo sapiens
1.5
-
cyclocreatine pH 9.0, 30°C, recombinant mutant I69L Homo sapiens
4.2
-
Creatine pH 9.0, 30°C, recombinant mutant I69A Homo sapiens
4.3
-
N-ethylglycocyamine pH 9.0, 30°C, recombinant mutant V325A Homo sapiens
4.4
-
cyclocreatine pH 9.0, 30°C, recombinant mutant I69V Homo sapiens
12
-
N-ethylglycocyamine pH 9.0, 30°C, recombinant mutant I69V Homo sapiens
14.5
-
N-ethylglycocyamine pH 9.0, 30°C, recombinant wild-type enzyme Homo sapiens
15.4
-
Creatine pH 9.0, 30°C, recombinant mutant V325A Homo sapiens
18
-
cyclocreatine pH 9.0, 30°C, recombinant mutant V325A Homo sapiens
35.2
-
cyclocreatine pH 9.0, 30°C, recombinant wild-type enzyme Homo sapiens
45
-
Creatine pH 9.0, 30°C, recombinant mutant I69L Homo sapiens
82.7
-
Creatine pH 9.0, 30°C, recombinant mutant I69V Homo sapiens
85.3
-
Creatine pH 9.0, 30°C, recombinant wild-type enzyme Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9
-
assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
MgADP-
-
Homo sapiens
MgATP2-
-
Homo sapiens