Cloned (Comment) | Organism |
---|---|
gene xdhA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain TP 1000 | Rhodobacter capsulatus |
Protein Variants | Comment | Organism |
---|---|---|
E232A | site-directed mutagenesis, the mutant exhibits a 12fold decrease in kred compared to wild-type | Rhodobacter capsulatus |
E232Q | site-directed mutagenesis, kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, the mutant exhibits a 12fold decrease in kred, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme | Rhodobacter capsulatus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | pH-dependent steady-state kinetics and reductive half-reaction, stopped flow kinetics, kinetic analysis of wild-type and mutant xanthine dehydrogenases, detailed overview. kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme. The ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of the substrate by two pH units and ensuring that at physiological pH the neutral form of the substrate predominates in the Michaelis complex. The product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release is not as great in the bacterial enzyme as compared with the vertebrate forms. The faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme | Rhodobacter capsulatus | |
0.114 | - |
xanthine | pH 8.5, 25°C, recombinant wild-type enzyme | Rhodobacter capsulatus | |
0.115 | - |
xanthine | pH 8.5, 25°c, recombinant mutant E232Q | Rhodobacter capsulatus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
xanthine + H2O + O2 | Rhodobacter capsulatus | - |
urate + H2O2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodobacter capsulatus | O54050 | small subunit encoded by gene xdhA | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and anion exchange chromatography, followed by gel filtration | Rhodobacter capsulatus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
xanthine + H2O + O2 = urate + H2O2 | mechanism of the reductive half-reaction of xanthine oxidase | Rhodobacter capsulatus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine. Proposed orientations of xanthine binding in the active site of xanthine oxidoreductase, using the predominant tautomers of the neutral and monoanionic forms of xanthine, overview | Rhodobacter capsulatus | ? | - |
? | |
xanthine + H2O + O2 | - |
Rhodobacter capsulatus | urate + H2O2 | - |
? |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Rhodobacter capsulatus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.14 | - |
xanthine | pH 8.5, 25°c, recombinant mutant E232Q | Rhodobacter capsulatus | |
135 | - |
xanthine | pH 8.5, 25°C, recombinant wild-type enzyme | Rhodobacter capsulatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
assay at | Rhodobacter capsulatus |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
additional information | - |
comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type enzyme and mutant E232Q, overview. The ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of the substrate by two pH units and ensuring that at physiological pH the neutral form of the substrate predominates in the Michaelis complex | Rhodobacter capsulatus |
General Information | Comment | Organism |
---|---|---|
additional information | enzyme active site structure, overview | Rhodobacter capsulatus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
18.6 | - |
xanthine | pH 8.5, 25°c, recombinant mutant E232Q | Rhodobacter capsulatus | |
1184.2 | - |
xanthine | pH 8.5, 25°C, recombinant wild-type enzyme | Rhodobacter capsulatus |