Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in Escherichia coli | Methanosarcina mazei |
Protein Variants | Comment | Organism |
---|---|---|
T307S | mutant reveals a 3.2fold improvement in kcat/Km for phosphothreonyl adenylate synthesis, as compared with wild-type SepRS. The mutant is unable to transfer phosphothreonine to tRNACys at greater than 10% plateau levels | Methanosarcina mazei |
T307S | site-directed mutagenesis, the mutant shows increased activity with phosphothreonine, thus reduced substrate specificity | Methanosarcina mazei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | recombinant enzyme, ATP/diphosphate and aminoacylation kinetics, Michaelis-Menten kinetics | Methanosarcina mazei | |
0.04 | - |
O-phospho-L-serine | wild-type enzyme | Methanosarcina mazei | |
0.15 | - |
O-phospho-L-serine | pH 7.5, 37°C, recombinant mutant T307S | Methanosarcina mazei | |
0.27 | - |
O-phospho-L-serine | pH 7.5, 37°C, recombinant enzyme | Methanosarcina mazei | |
0.93 | - |
O-phospho-L-threonine | pH 7.5, 37°C, recombinant mutant T307S | Methanosarcina mazei | |
2.2 | - |
O-phospho-L-threonine | pH 7.5, 37°C, recombinant enzyme | Methanosarcina mazei | |
8.4 | - |
O-phospho-L-threonine | wild-type enzyme | Methanosarcina mazei |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Methanosarcina mazei |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
60909 | - |
4 * 60909, calculated from sequence | Methanosarcina mazei |
60909 | - |
4 * 60909, sequence calculation, 4 * 68992, homotetramer alpha4, mass spectrometry | Methanosarcina mazei |
68992 | - |
4 * 60909, sequence calculation, 4 * 68992, homotetramer alpha4, mass spectrometry | Methanosarcina mazei |
250000 | - |
gel filtration | Methanosarcina mazei |
250000 | - |
recombinant enzyme,native PAGE, and gel filtration | Methanosarcina mazei |
255400 | - |
recombinant enzyme, mass spectrometry | Methanosarcina mazei |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + O-phospho-L-serine + tRNACys | Methanosarcina mazei | - |
AMP + diphosphate + O-phospho-L-seryl-tRNACys | - |
? | |
ATP + O-phospho-L-serine + tRNACys | Methanosarcina mazei | half-of-the-sites activity: the tetrameric enzyme binds two tRNAs. Only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate | AMP + diphosphate + O-phospho-L-seryl-tRNACys | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Methanosarcina mazei | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant | Methanosarcina mazei |
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli to homogeneity by nickel affinity chromatography, the His-tag is cleaved off | Methanosarcina mazei |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
recombinant enzyme, ATP-diphosphate exchange activity | Methanosarcina mazei |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + O-phospho-L-serine + tRNACys | - |
Methanosarcina mazei | AMP + diphosphate + O-phospho-L-seryl-tRNACys | - |
? | |
ATP + O-phospho-L-serine + tRNACys | half-of-the-sites activity: the tetrameric enzyme binds two tRNAs. Only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate | Methanosarcina mazei | AMP + diphosphate + O-phospho-L-seryl-tRNACys | - |
? | |
ATP + O-phospho-L-serine + tRNACys | half-of-the-sites activity: the tetrameric enzyme binds two tRNAs. Only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Efficient phosphoserylation by SepRS requires methylation of tRNACys at the N1 position of G37 in the anticodon loop | Methanosarcina mazei | AMP + diphosphate + O-phospho-L-seryl-tRNACys | - |
? | |
ATP + O-phospho-L-serine + tRNACys | the tetrameric enzyme binds two tRNAs and only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. tRNACys binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme | Methanosarcina mazei | AMP + diphosphate + O-phospho-L-seryl-tRNACys | - |
? | |
ATP + O-phospho-L-threonine + tRNACys | low activity | Methanosarcina mazei | AMP + diphosphate + O-phospho-L-threonyl-tRNACys | - |
? | |
ATP + O-phospho-L-threonine + tRNACys | about 35% of the plateau aminoacylation observed with O-phospho-L-serine | Methanosarcina mazei | AMP + diphosphate + O-phospho-L-threonyl-tRNACys | - |
? | |
additional information | SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site | Methanosarcina mazei | ? | - |
? | |
additional information | SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. Determination of the ATP-diphosphate exchange activity. Serine and glutamate are poor substrates, overview | Methanosarcina mazei | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the tetrameric enzyme binds two tRNAs and only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate, active site titrations reveal calculation of 2.3 active sites per tetramer, overview | Methanosarcina mazei |
tetramer | 4 * 60909, calculated from sequence | Methanosarcina mazei |
tetramer | 4 * 60909, sequence calculation, 4 * 68992, homotetramer alpha4, mass spectrometry | Methanosarcina mazei |
Synonyms | Comment | Organism |
---|---|---|
More | the enzyme is a class II tRNA synthetase | Methanosarcina mazei |
phosphoseryl-tRNA synthetase | - |
Methanosarcina mazei |
SepRS | - |
Methanosarcina mazei |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Methanosarcina mazei |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0054 | - |
O-phospho-L-threonine | wild-type enzyme | Methanosarcina mazei | |
0.014 | - |
O-phospho-L-threonine | pH 7.5, 37°C, recombinant enzyme | Methanosarcina mazei | |
0.021 | - |
O-phospho-L-threonine | pH 7.5, 37°C, recombinant mutant T307S | Methanosarcina mazei | |
0.45 | - |
O-phospho-L-serine | wild-type enzyme | Methanosarcina mazei | |
10.6 | - |
O-phospho-L-serine | pH 7.5, 37°C, recombinant enzyme | Methanosarcina mazei | |
11.2 | - |
O-phospho-L-serine | pH 7.5, 37°C, recombinant mutant T307S | Methanosarcina mazei |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Methanosarcina mazei |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Methanosarcina mazei |