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Literature summary for 2.7.7.12 extracted from
- 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.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| overexpression of mutants Q168N and Q168R in strain Bl21(DE3) | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| Q168N | site-directed mutagenesis, 50fold reduced activity compared to wild-type, 40fold decreased kcat | Escherichia coli |
| 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]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetics | Escherichia coli | |
| additional information | - |
additional information | kinetics at 4°C, wild-type and mutants | Escherichia coli | |
| additional information | - |
additional information | wild-type and mutants in the 2-step reaction | Escherichia coli |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Fe2+ | required | Escherichia coli |  |
| Fe2+ | contains 0.67 mol per mol of wild-type enzyme subunit, 0.44 mol per mol of mutant Q168R subunit | Escherichia coli | |
| Zn2+ | required | Escherichia coli | |
| Zn2+ | 1.2 mol tightly bound per mol of subunit | Escherichia coli | |
| Zn2+ | 0.91 mol per mol of mutant Q168R subunit | Escherichia coli | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant from overexpressing strain BL21(DE3) | Escherichia coli |
| recombinant wild-type and mutants Q168R and Q168N | Escherichia coli |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| 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 |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.00066 | - |
purified recombinant mutant Q168R | Escherichia coli |
| 3.4 | - |
purified recombinant mutant Q168N | Escherichia coli |
| 180 | - |
purified recombinant wild-type enzyme | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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 | |
| 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]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 27 | - |
assay at | Escherichia coli |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | - |
Escherichia coli | |
| 281 | - |
alpha-D-galactose 1-phosphate | recombinant wild-type, pH 8.5, 4°C | Escherichia coli | |
| 780 | - |
alpha-D-galactose 1-phosphate | recombinant wild-type, forward reaction, pH 8.5, 27°C | Escherichia coli | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8.5 | - |
assay at | Escherichia coli |
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