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Literature summary for 5.4.99.2 extracted from

  • Makins, C.; Pickering, A.V.; Mariani, C.; Wolthers, K.R.
    Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase (2013), Biochemistry, 52, 878-888.
    View publication on PubMed
Search for more literature for 5.4.99.2

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta(DE3)pLysS cells Homo sapiens

Protein Variants

Protein Variants Comment Organism
E392A site-directed mutagenesis, kcat is reduced 12fold compared to the wild-type enzyme. The mutant shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate Homo sapiens
E392D site-directed mutagenesis, kcat is reduced 330fold compared to the wild-type enzyme. The mutant shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate Homo sapiens
E392Q site-directed mutagenesis, kcat is reduced 16fold compared to the wild-type enzyme. The mutant shows no detectable adenosylcobalamin homolysis upon binding of the physiological substrate Homo sapiens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information pre-steady-state and steady-state kinetics of wild-type and mutant enzymes Homo sapiens

Localization

Localization Comment Organism GeneOntology No. Textmining
mitochondrion
-
 Homo sapiens 5739
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(R)-methylmalonyl-CoA Homo sapiens
-
 succinyl-CoA
-
 ?

Organism

Organism UniProt Comment Textmining
Homo sapiens P22033
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta(DE3)pLysS cells by nickel affinity and anion exchange chromatography Homo sapiens

Reaction

Reaction Comment Organism Reaction ID
(R)-methylmalonyl-CoA = succinyl-CoA mechanism, overview, a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for Co-C bond homolysis, key role for the conserved polar glutamate residue in controlling the initial generation of radical species Homo sapiens
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-methylmalonyl-CoA
-
 Homo sapiens succinyl-CoA
-
 ?
(R)-methylmalonyl-CoA binding of substrate to the enzyme leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin cofactor. Residue Glu392 is involved in adenosylcobalamin Co-C bond labilization and catalysis Homo sapiens succinyl-CoA
-
 ?

Synonyms

Synonyms Comment Organism
MCM
-
 Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.012
-
 (R)-methylmalonyl-CoA recombinant His-tagged mutant E392D, pH 8.5, 25°C Homo sapiens
0.25
-
 (R)-methylmalonyl-CoA recombinant His-tagged mutant E392Q, pH 8.5, 25°C Homo sapiens
0.32
-
 (R)-methylmalonyl-CoA recombinant His-tagged mutant E392A, pH 8.5, 25°C Homo sapiens
3.95
-
 (R)-methylmalonyl-CoA recombinant His-tagged wild-type enzyme, pH 8.5, 25°C Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
 assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
adenosylcobalamin binding analysis with recombinant wild-type and mutant enzymes, overview Homo sapiens