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Literature summary for 2.7.3.3 extracted from

  • Michibata, J.; Okazaki, N.; Motomura, S.; Uda, K.; Fujiwara, S.; Suzuki, T.
    Two arginine kinases of Tetrahymena pyriformis: characterization and localization (2014), Comp. Biochem. Physiol. B, 171, 34-41.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene AK1, DNA and amino acid sequence determination and analysis, recombinant expression of C-terminally His6-tagged isozyme AK1 in Escherichia coli strain BL21 (DE3). The prokaryotic protein expression host Escherichia coli uses the standard genetic code while Tetrahymena pyriformis uses an alternative genetic code where TAA and TAG code for glutamine instead of a stop codon, therefore a TAA codon in AK1 is replaced with CAA Tetrahymena pyriformis
gene AK2, DNA and amino acid sequence determination and analysis, recombinant expression of C-terminally His6-tagged isozyme AK2 in Escherichia coli strain BL21(DE3). The prokaryotic protein expression host Escherichia coli uses the standard genetic code while Tetrahymena pyriformis uses an alternative genetic code where TAA and TAG code for glutamine instead of a stop codon., therefore two TAA and three TAG codons in AK2 are replaced with CAA and CAG Tetrahymena pyriformis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetic analysis, overview. The enzyme shows a random-order, rapid equilibrium kinetic mechanism Tetrahymena pyriformis
0.26
-
L-arginine pH 8.0, 25°C, recombinant His6-tagged isozyme AK1 Tetrahymena pyriformis
0.35
-
L-arginine pH 8.0, 25°C, recombinant His6-tagged isozyme AK2 Tetrahymena pyriformis
0.73
-
ATP pH 8.0, 25°C, recombinant His6-tagged isozyme AK1 Tetrahymena pyriformis
0.73
-
ATP pH 8.0, 25°C, recombinant His6-tagged isozyme AK2 Tetrahymena pyriformis

Localization

Localization Comment Organism GeneOntology No. Textmining
cytoplasm
-
Tetrahymena pyriformis 5737
-
membrane the amino acid sequence of Tetrahymena pyriformis isozyme AK1, but not isozyme AK2, has a distinct myristoylation signal sequence at the N-terminus, suggesting that 40-kDa AK1 targets the membrane Tetrahymena pyriformis 16020
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Tetrahymena pyriformis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
40000
-
-
Tetrahymena pyriformis
80000
-
-
Tetrahymena pyriformis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-arginine Tetrahymena pyriformis
-
ADP + Nomega-phospho-L-arginine
-
r

Organism

Organism UniProt Comment Textmining
Tetrahymena pyriformis U3T1K4 isozyme AK1
-
Tetrahymena pyriformis U3T2Y2 isozyme AK2
-

Posttranslational Modification

Posttranslational Modification Comment Organism
lipoprotein isozyme AK1 has a distinct myristoylation signal sequence at the N-terminus Tetrahymena pyriformis

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His6-tagged isozyme AK1 from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography Tetrahymena pyriformis

Reaction

Reaction Comment Organism Reaction ID
ATP + L-arginine = ADP + Nomega-phospho-L-arginine random-order, rapid equilibrium kinetic mechanism Tetrahymena pyriformis

Source Tissue

Source Tissue Comment Organism Textmining
flagellum
-
Tetrahymena pyriformis
-
additional information isozyme AK1 is mainly localized in the ciliary fraction, AK1 is attached to the ciliary membrane through the myristoylation group Tetrahymena pyriformis
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-arginine
-
Tetrahymena pyriformis ADP + Nomega-phospho-L-arginine
-
r

Subunits

Subunits Comment Organism
? x * 40000, isozyme AK1, SDS-PAGE Tetrahymena pyriformis
? x * 80000, isozyme AK2, SDS-PAGE Tetrahymena pyriformis

Synonyms

Synonyms Comment Organism
AK1
-
Tetrahymena pyriformis
AK2
-
Tetrahymena pyriformis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Tetrahymena pyriformis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
63.4
-
ATP pH 8.0, 25°C, recombinant His6-tagged isozyme AK2 Tetrahymena pyriformis
63.4
-
L-arginine pH 8.0, 25°C, recombinant His6-tagged isozyme AK2 Tetrahymena pyriformis
104
-
ATP pH 8.0, 25°C, recombinant His6-tagged isozyme AK1 Tetrahymena pyriformis
104
-
L-arginine pH 8.0, 25°C, recombinant His6-tagged isozyme AK1 Tetrahymena pyriformis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Tetrahymena pyriformis

Cofactor

Cofactor Comment Organism Structure
ADP
-
Tetrahymena pyriformis
ATP
-
Tetrahymena pyriformis

General Information

General Information Comment Organism
evolution two putative AK genes in the genome of Tetrahymena thermophila: one is a typical AK with a 40-kDa subunit (AK1) and the other is an unusual two-domain AK2 having an 80-kDa contiguous dimer, which appears to be the result of gene duplication and subsequent fusion Tetrahymena pyriformis
additional information the five residues S63, Y68, E225, C271, and E314 that interact with the arginine substrate are conserved, as well as the five Arg residues R124, R126, R229, R280 and R309 that interact with substrate ATP. Residues D62 and R193 are suggested to play a key role in stabilizing the substrate-bound structures of AK by forming an ion pair. Tyr89 is also a key residue in typical invertebrate AKs and is strictly conserved. This residue is not directly involved in substrate binding but it is located close to the site that bindswith the substrate arginine, it significantly and specifically affects guanidino substrate Tetrahymena pyriformis
physiological function isozyme AK1 plays a role in the phosphoarginine shuttle, which enables a continuous energy flow to dynein for ciliary movement in Tetrahymena pyriformis Tetrahymena pyriformis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
86.9
-
ATP pH 8.0, 25°C, recombinant His6-tagged isozyme AK2 Tetrahymena pyriformis
142.5
-
ATP pH 8.0, 25°C, recombinant His6-tagged isozyme AK1 Tetrahymena pyriformis
181.1
-
L-arginine pH 8.0, 25°C, recombinant His6-tagged isozyme AK2 Tetrahymena pyriformis
400
-
L-arginine pH 8.0, 25°C, recombinant His6-tagged isozyme AK1 Tetrahymena pyriformis