Activating Compound | Comment | Organism | Structure |
---|---|---|---|
orotidine 5'-phosphate | the 5'-phosphate group of the substrate activates the enzyme 240000000fold. The binding of orotidine 5'-monophosphate is accompanied by a conformational change of the enzyme from an open, inactive conformation to a closed, active conformation. As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions that destabilize the carboxylate group of the substrate and stabilize the intermediate are enforced. The activation is equivalently described by an intrinsic binding energy of 11.4 kcal/mol. The residues that directly contact the 5'-phosphate group, participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from the solvent, or that form hydrogen bonding interactions across the interface between the mobile and fixed half-barrel domains of the (beta/alpha)8-barrel structure. The data support a model in which the intrinsic binding energy provided by the 5'-phosphate group is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize the complexes of the enzyme in the active closed conformation with the substrate or with the substrate intermediate with the destabilized carboxylate group relative to the complex of the enzyme in the open inactive conformation with the substrate | Methanothermobacter thermautotrophicus |
Crystallization (Comment) | Organism |
---|---|
eight different crystal forms are grown by the sitting drop method at room temperature for single mutant enzymes liganded with 1-(5-phospho-beta-ribofuranosyl)barbituric acid: Q185A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], R203A/[1-(5-phospho-beta-ribofuranosyl)barbituric acid], T159V/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], T159A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], T159S/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], R160A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid], Y206F/[1-(5-phospho-beta-ribofuranosyl)barbituric acid], K82A/[1-(5-phospho-beta-D-ribofuranosyl)barbituric acid] | Methanothermobacter thermautotrophicus |
mutants Q185A, R203A, T159V, T159A, T159S, R160A, Y206F, K82A, and double- and triple mutants, to 1.3-1.6 A resolution | Methanothermobacter thermautotrophicus |
Protein Variants | Comment | Organism |
---|---|---|
K82A | crystallization data | Methanothermobacter thermautotrophicus |
Q185A | 220fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
Q185A/R203A | loss of activity | Methanothermobacter thermautotrophicus |
R160A | 43fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
R160A/R203A | loss of activity | Methanothermobacter thermautotrophicus |
R160A/V182A | 1700fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
R160A/Y206F | crystallization data | Methanothermobacter thermautotrophicus |
R160A/Y206F | 340fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
R203A | 1900fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
R203A/R160A | crystallization data | Methanothermobacter thermautotrophicus |
R203A/T159V | crystallization data | Methanothermobacter thermautotrophicus |
R203A/V182A | crystallization data | Methanothermobacter thermautotrophicus |
T159A | crystallization data | Methanothermobacter thermautotrophicus |
T159S | crystallization data | Methanothermobacter thermautotrophicus |
T159V | 41fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
T159V/R203A | loss of activity | Methanothermobacter thermautotrophicus |
T159V/V182A | 3600fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
T159V/V182A/Y206F | 20000fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
T159V/Y206F | 780fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
V182A | 22fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
V182A/R203A | loss of activity | Methanothermobacter thermautotrophicus |
V182A/Y206F | 190fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
Y206F | 4fold decrease in catalytic efficiency | Methanothermobacter thermautotrophicus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0018 | - |
orotidine 5'-phosphate | wild-type, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.0055 | - |
orotidine 5'-phosphate | mutant Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.024 | - |
orotidine 5'-phosphate | mutant V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.03 | - |
orotidine 5'-phosphate | mutant R160A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.033 | - |
orotidine 5'-phosphate | mutant T159V, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.1 | - |
orotidine 5'-phosphate | mutant V182A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.1 | 1 | orotidine 5'-phosphate | mutant Q185A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.13 | - |
orotidine 5'-phosphate | mutant R160A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.3 | - |
orotidine 5'-phosphate | mutant T159V/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.6 | - |
orotidine 5'-phosphate | mutant R160A/V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.7 | - |
orotidine 5'-phosphate | mutant T159V/V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.98 | - |
orotidine 5'-phosphate | mutant R203A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.9 | - |
orotidine 5'-phosphate | mutant T159V/V182A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Methanothermobacter thermautotrophicus | O26232 | - |
- |
Methanothermobacter thermautotrophicus DSM 1053 | O26232 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
Orotidine 5'-phosphate | the remote 5'-phosphate group of the substrate activates the enzyme 240 millionfold, the activation corresponds to an intrinsic binding energy of 11.4 kcal/mol. This intrinsic binding energy is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec-S and Ec-S* complexes relative to the Eo-S complex | Methanothermobacter thermautotrophicus | UMP + CO2 | - |
? | |
Orotidine 5'-phosphate | the remote 5'-phosphate group of the substrate activates the enzyme 240 millionfold, the activation corresponds to an intrinsic binding energy of 11.4 kcal/mol. This intrinsic binding energy is used to allow interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec-S and Ec-S* complexes relative to the Eo-S complex | Methanothermobacter thermautotrophicus DSM 1053 | UMP + CO2 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
OMPDC | - |
Methanothermobacter thermautotrophicus |
Orotidine 5'-monophosphate decarboxylase | - |
Methanothermobacter thermautotrophicus |
PyrF | - |
Methanothermobacter thermautotrophicus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Methanothermobacter thermautotrophicus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.24 | - |
orotidine 5'-phosphate | mutant T159V/V182A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.56 | - |
orotidine 5'-phosphate | mutant T159V/V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1 | - |
orotidine 5'-phosphate | mutant R160A/V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.1 | - |
orotidine 5'-phosphate | mutant R160A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.1 | - |
orotidine 5'-phosphate | mutant T159V/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.4 | - |
orotidine 5'-phosphate | mutant Q185A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.5 | - |
orotidine 5'-phosphate | mutant R203A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.5 | - |
orotidine 5'-phosphate | mutant V182A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
2 | - |
orotidine 5'-phosphate | mutant R160A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
2.3 | - |
orotidine 5'-phosphate | mutant T159V, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
3 | - |
orotidine 5'-phosphate | mutant V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
4.1 | - |
orotidine 5'-phosphate | mutant Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
5.3 | - |
orotidine 5'-phosphate | wild-type, pH 7.1, 25°C | Methanothermobacter thermautotrophicus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.1 | - |
assay at | Methanothermobacter thermautotrophicus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.13 | - |
orotidine 5'-phosphate | mutant T159V/V182A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
0.8 | - |
orotidine 5'-phosphate | mutant T159V/V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.5 | - |
orotidine 5'-phosphate | mutant R203A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
1.7 | - |
orotidine 5'-phosphate | mutant R160A/V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
3.7 | - |
orotidine 5'-phosphate | mutant T159V/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
8.5 | - |
orotidine 5'-phosphate | mutant R160A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
13 | - |
orotidine 5'-phosphate | mutant Q185A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
15 | - |
orotidine 5'-phosphate | mutant V182A/Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
67 | - |
orotidine 5'-phosphate | mutant R160A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
70 | - |
orotidine 5'-phosphate | mutant T159V, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
130 | - |
orotidine 5'-phosphate | mutant V182A, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
750 | - |
orotidine 5'-phosphate | mutant Y206F, pH 7.1, 25°C | Methanothermobacter thermautotrophicus | |
2900 | - |
orotidine 5'-phosphate | wild-type, pH 7.1, 25°C | Methanothermobacter thermautotrophicus |