Cloned (Comment) | Organism |
---|---|
gene purT, recombinant expression of wild-type and mutant enzymes in TX680 auxotrophic cells | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
G162I | site-directed mutagenesis, the enzyme mutant releases formyl phosphate into solution, reduced activity compared to wild-type enzyme | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics | Escherichia coli | |
0.0101 | - |
N1-(5-phospho-beta-D-ribosyl)glycinamide | recombinant enzyme, pH 8.0, 25°C | Escherichia coli | |
0.036 | - |
ADP | recombinant enzyme, pH 8.0, 25°C, reaction with formyl phosphate | Escherichia coli | |
0.045 | - |
ATP | recombinant enzyme, pH 8.0, 25°C, with formate | Escherichia coli | |
0.077 | - |
ATP | recombinant enzyme, pH 8.0, 25°C, with acetate | Escherichia coli | |
0.26 | - |
ADP | recombinant enzyme, pH 8.0, 25°C, reaction with acetyl phosphate | Escherichia coli | |
0.319 | - |
formate | recombinant enzyme, pH 8.0, 25°C | Escherichia coli | |
0.48 | - |
acetyl phosphate | recombinant enzyme, pH 8.0, 25°C, reaction with ADP | Escherichia coli | |
0.49 | - |
ADP | recombinant enzyme, pH 8.0, 25°C, reaction with carbamoyl phosphate | Escherichia coli | |
1.17 | - |
Carbamoyl phosphate | recombinant enzyme, pH 8.0, 25°C, reaction with ADP | Escherichia coli | |
1.3 | - |
formyl phosphate | recombinant enzyme, pH 8.0, 25°C, reaction with ADP | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + formate + N1-(5-phospho-beta-D-ribosyl)glycinamide | Escherichia coli | - |
ADP + phosphate + N2-formyl-N1-(5-phospho-beta-D-ribosyl)glycinamide | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P33221 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from TX680 auxotrophic cells by gel filtration, anion exchange chromatography, and dialysis, to over 90% purity | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ADP + acetyl phosphate | only half-reaction | Escherichia coli | ? | - |
? | |
ADP + carbamoyl phosphate | only half-reaction | Escherichia coli | ? | - |
? | |
ADP + formyl phosphate | only half-reaction | Escherichia coli | ? | - |
? | |
ATP + acetate + N1-(5-phospho-beta-D-ribosyl)glycinamide | only half-reaction resulting in formation of ADP and acetylphosphate | Escherichia coli | ? | - |
? | |
ATP + formate + N1-(5-phospho-beta-D-ribosyl)glycinamide | - |
Escherichia coli | ADP + phosphate + N2-formyl-N1-(5-phospho-beta-D-ribosyl)glycinamide | - |
? | |
additional information | kinetic studies of the wild-type PurT enzyme demonstrate that formyl phosphate behaves as a chemically and kinetically competent intermediate. The requirement for ATP and glycinamide ribonucleotide (GAR) in these reactions is consistent with previous steady-state kinetic results, which have demonstrated that all substrates must be bound before catalysis. Kinetic analysis and positional isotope exchange studies also support the assignment of formyl phosphate as a plausible intermediate. Forward and reverse half-reactions utilizing the proposed intermediate are measured spectrophotometrically. PurT transformylase is capable of generating acetyl phosphate from ATP and acetate. The first half-reaction leads to production of acyl phosphate, the second half-reaction acylates GAR. Neither acetyl GAR nor carbamyl GAR is produced when the enzyme is incubated with ATP, GAR, and the appropriate acid. Neither the reverse first half-reaction with acetyl phosphate nor carbamyl phosphate requires GAR to be present, in contrast to the half-reactions involving formyl phosphate. No activity with aminoformate | Escherichia coli | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
GAR transformylase | - |
Escherichia coli |
glycinamide ribonucleotide transformylase | - |
Escherichia coli |
PurT | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | The purT encoded glycinamide ribonucleotide transformylase differs from the previously known purN encoded enzyme in size, sequence, and substrates, and ATP and formate are required as opposed to formyl tetrahydrofolate | Escherichia coli |
malfunction | PurT transformylase mutant G162I catalyzes the production of formyl GAR two orders of magnitude less efficiently than the wild-type enzyme. This reduced rate is apparently sufficient to sustain cell growth under limiting purine conditions | Escherichia coli |
physiological function | the Escherichia coli purT encoded glycinamide ribonucleotide transformylase (GAR transformylase) serves as an alternate enzyme in the production of formyl GAR for use in de novo purine biosynthesis | Escherichia coli |