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Literature summary extracted from

  • Geeganage, S.; Frey, P.A.
    Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia (1998), Biochemistry, 37, 14500-14507.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.12 overexpression of mutants Q168N and Q168R in strain Bl21(DE3) Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
2.7.7.12 Q168N site-directed mutagenesis, 50fold reduced activity compared to wild-type, 40fold decreased kcat Escherichia coli
2.7.7.12 Q168R site-directed mutagenesis, 270000fold reduced activity compared to wild-type, i.e. nearly no remaining activity, mutant active sites can be uridylated by 65%, with very slow deuridylylation, compared to 100% for the wild-type, reduced metal content Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.7.12 additional information
-
 additional information kinetics Escherichia coli
2.7.7.12 additional information
-
 additional information kinetics at 4°C, wild-type and mutants Escherichia coli
2.7.7.12 additional information
-
 additional information wild-type and mutants in the 2-step reaction Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.7.12 Fe2+ required Escherichia coli
2.7.7.12 Fe2+ contains 0.67 mol per mol of wild-type enzyme subunit, 0.44 mol per mol of mutant Q168R subunit Escherichia coli
2.7.7.12 Zn2+ required Escherichia coli
2.7.7.12 Zn2+ 1.2 mol tightly bound per mol of subunit Escherichia coli
2.7.7.12 Zn2+ 0.91 mol per mol of mutant Q168R subunit Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.7.12 UDP-glucose + alpha-D-galactose 1-phosphate Escherichia coli involved in conversion of galactose into alpha-D-glucose 1-phosphate, Leloir pathway alpha-D-glucose 1-phosphate + UDP-galactose
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.12 Escherichia coli
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.12 recombinant from overexpressing strain BL21(DE3) Escherichia coli
2.7.7.12 recombinant wild-type and mutants Q168R and Q168N Escherichia coli

Reaction

EC Number Reaction Comment Organism Reaction ID
2.7.7.12 UDP-alpha-D-glucose + alpha-D-galactose 1-phosphate = alpha-D-glucose 1-phosphate + UDP-alpha-D-galactose Gln168 engages hydrogen bonding with the phosphoryl oxygen of the UMP moiety in the covalently formed reaction intermediate UMP-enzyme, UMP is bound to His166 Escherichia coli
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.7.7.12 0.00066
-
 purified recombinant mutant Q168R Escherichia coli
2.7.7.12 3.4
-
 purified recombinant mutant Q168N Escherichia coli
2.7.7.12 180
-
 purified recombinant wild-type enzyme Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.12 UDP-glucose + alpha-D-galactose 1-phosphate formation of a covalent uridylyl-enzyme intermediate, i.e. UMP-enzyme Escherichia coli alpha-D-glucose 1-phosphate + UDP-galactose
-
 r
2.7.7.12 UDP-glucose + alpha-D-galactose 1-phosphate involved in conversion of galactose into alpha-D-glucose 1-phosphate, Leloir pathway Escherichia coli alpha-D-glucose 1-phosphate + UDP-galactose
-
 r

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.7.12 27
-
 assay at Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.7.12 additional information
-
 additional information
-
 Escherichia coli
2.7.7.12 281
-
 alpha-D-galactose 1-phosphate recombinant wild-type, pH 8.5, 4°C Escherichia coli
2.7.7.12 780
-
 alpha-D-galactose 1-phosphate recombinant wild-type, forward reaction, pH 8.5, 27°C Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.7.12 8.5
-
 assay at Escherichia coli