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Literature summary extracted from

  • Lovelace, L.L.; Johnson, S.R.; Gibson, L.M.; Bell, B.J.; Berger, S.H.; Lebioda, L.
    Variants of human thymidylate synthase with loop 181-197 stabilized in the inactive conformation (2009), Protein Sci., 18, 1628-1636.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.45 expressed in Escherichia coli strain TX61 Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.45 hanging drop vapor diffusion method, using 2.0 M ammonium sulfate, 0.1 M Tris, pH 8.5, and 20 mM beta-mercaptoethanol Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.45 A191K the mutant enzyme displays a kcat value of 6fold lower than wild type Homo sapiens
2.1.1.45 L198P the mutant enzyme displays a kcat value of 2fold lower than wild type Homo sapiens
2.1.1.45 M190E the mutant enzyme displays a kcat value of 5.9fold lower than wild type Homo sapiens
2.1.1.45 M190K the mutant has the value of kcat/Km smaller by a factor of about 7500 than the wild type, the crystal structure of this mutant is similar to that of the wild type with loop 181-197 in the inactive conformation, however, the direct vicinity of the mutation, residues 188-194 of this loop, assumes a different conformation with the positions of Calpha shifted up to 7.2 A, the mutant protein is trapped in an inactive state that does not equilibrate easily with the active conformer Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.45 0.00085
-
 dUMP mutant enzyme A191K, at 37°C Homo sapiens
2.1.1.45 0.00096
-
 dUMP mutant enzyme M190E, at 37°C Homo sapiens
2.1.1.45 0.00259
-
 dUMP wild type enzyme, at 37°C Homo sapiens
2.1.1.45 0.01563
-
 dUMP mutant enzyme L198P, at 37°C Homo sapiens
2.1.1.45 0.179
-
 dUMP mutant enzyme M190K, at 37°C Homo sapiens

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.45 Homo sapiens P04818
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.45 Blue-Sepharose column chromatography and Q-Sepharose column chromatography Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.45 5,10-methylenetetrahydrofolate + dUMP
-
 Homo sapiens dihydrofolate + dTMP
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.1.45 0.03
-
 dUMP mutant enzyme M190K, at 37°C Homo sapiens
2.1.1.45 0.52
-
 dUMP mutant enzyme A191K, at 37°C Homo sapiens
2.1.1.45 0.53
-
 dUMP mutant enzyme M190E, at 37°C Homo sapiens
2.1.1.45 1.64
-
 dUMP mutant enzyme L198P, at 37°C Homo sapiens
2.1.1.45 3.11
-
 dUMP wild type enzyme, at 37°C Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.45 N5,N10-methylenetetrahydrofolate
-
 Homo sapiens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.1.1.45 0.00000016
-
 dUMP mutant enzyme M190K, at 37°C Homo sapiens
2.1.1.45 0.0001
-
 dUMP mutant enzyme L198P, at 37°C Homo sapiens
2.1.1.45 0.00055
-
 dUMP mutant enzyme M190E, at 37°C Homo sapiens
2.1.1.45 0.00061
-
 dUMP mutant enzyme A191K, at 37°C Homo sapiens
2.1.1.45 0.0012
-
 dUMP wild type enzyme, at 37°C Homo sapiens