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Literature summary for 5.3.3.2 extracted from

  • Nagai, T.; Unno, H.; Janczak, M.W.; Yoshimura, T.; Poulter, C.D.; Hemmi, H.
    Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors (2011), Proc. Natl. Acad. Sci. USA, 108, 20461-20466.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5. The high-resolution crystal structures of enzyme-substrate complexes and the kinetic studies of new mutants confirm that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer Saccharolobus shibatae

Protein Variants

Protein Variants Comment Organism
Q160E 10-fold decrease in kcat/Km Saccharolobus shibatae
Q160E the mutant shows a 10fold decrease in kcat/Km Saccharolobus shibatae
Q160H 23-fold decrease in kcat/Km Saccharolobus shibatae
Q160H the mutant shows a 23fold decrease in kcat/Km Saccharolobus shibatae
Q160K 130-fold decrease in kcat/Km Saccharolobus shibatae
Q160K the mutant shows a 130fold decrease in kcat/Km Saccharolobus shibatae
Q160L 28-fold decrease in kcat/Km Saccharolobus shibatae
Q160L the mutant shows a 28fold decrease in kcat/Km Saccharolobus shibatae
Q160N 150-fold decrease in kcat, kcat/Km decreases 66-fold Saccharolobus shibatae
Q160N the mutant shows a 150fold decrease in kcat, although kcat/Km only decreases 66fold Saccharolobus shibatae

Inhibitors

Inhibitors Comment Organism Structure
3-methylene-4-penten-1-yl diphosphate irreversible inhibition; the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5 Saccharolobus shibatae
3-oxiranyl-3-buten-1-yl diphosphate irreversible inhibition; the covalent adduct formed between irreversible mechanism based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor are investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of enzyme binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5 Saccharolobus shibatae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0015
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160L Saccharolobus shibatae
0.00152
-
isopentenyl diphosphate mutant enzyme Q160L, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.0032
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160N Saccharolobus shibatae
0.00323
-
isopentenyl diphosphate mutant enzyme Q160N, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.0071
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160H Saccharolobus shibatae
0.00713
-
isopentenyl diphosphate mutant enzyme Q160H, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.00739
-
isopentenyl diphosphate wild type enzyme, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.0074
-
isopentenyl diphosphate pH 6.0, 60°C, wild-type enzyme Saccharolobus shibatae
0.00826
-
isopentenyl diphosphate mutant enzyme Q160E, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.0083
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160E Saccharolobus shibatae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
isopentenyl diphosphate Saccharolobus shibatae
-
dimethylallyl diphosphate
-
?

Organism

Organism UniProt Comment Textmining
Saccharolobus shibatae
-
-
-
Saccharolobus shibatae P61615
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
isopentenyl diphosphate
-
Saccharolobus shibatae dimethylallyl diphosphate
-
?
isopentenyl diphosphate a mechanism is proposed where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation Saccharolobus shibatae dimethylallyl diphosphate
-
?

Synonyms

Synonyms Comment Organism
Idi-2
-
Saccharolobus shibatae
isopentenyl diphosphate isomerase
-
Saccharolobus shibatae
type-2 isopentenyl diphosphate isomerase
-
Saccharolobus shibatae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
assay at Saccharolobus shibatae

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.000197
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160N Saccharolobus shibatae
0.00022
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160L Saccharolobus shibatae
0.001
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160H Saccharolobus shibatae
0.003
-
isopentenyl diphosphate pH 6.0, 60°C, mutant enzyme Q160E Saccharolobus shibatae
0.03
-
isopentenyl diphosphate pH 6.0, 60°C, wild-type enzyme Saccharolobus shibatae
197
-
isopentenyl diphosphate mutant enzyme Q160N, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
221
-
isopentenyl diphosphate mutant enzyme Q160L, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
1210
-
isopentenyl diphosphate mutant enzyme Q160H, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
3280
-
isopentenyl diphosphate mutant enzyme Q160E, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
29900
-
isopentenyl diphosphate wild type enzyme, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
assay at Saccharolobus shibatae

Cofactor

Cofactor Comment Organism Structure
FMN a mechanism is proposed where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation Saccharolobus shibatae

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.031
-
isopentenyl diphosphate mutant enzyme Q160K, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.061
-
isopentenyl diphosphate mutant enzyme Q160N, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.145
-
isopentenyl diphosphate mutant enzyme Q160L, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.17
-
isopentenyl diphosphate mutant enzyme Q160H, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
0.397
-
isopentenyl diphosphate mutant enzyme Q160E, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae
4.05
-
isopentenyl diphosphate wild type enzyme, in 50 mM MOPS-NaOH, pH 6.0, at 60°C Saccharolobus shibatae