Cloned (Comment) | Organism |
---|---|
gene XDH1, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli | 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 | steady-state kinetics and reductive half-reaction, stopped flow kinetics, kinetic analysis of wild-type and mutant xanthine dehydrogenases, 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 isdue to a faster rate constant for product release than is seen with the vertebrate enzyme | Rhodobacter capsulatus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
xanthine + NAD+ + H2O | Rhodobacter capsulatus | - |
urate + NADH + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodobacter capsulatus | O54050 AND O54051 | small and large subunits encoded by genes xdhA and xdhB | - |
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 |
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 | Rhodobacter capsulatus | ? | - |
? | |
xanthine + NAD+ + H2O | - |
Rhodobacter capsulatus | urate + NADH + H+ | - |
? | |
xanthine + NAD+ + H2O | product release is principally rate-limiting in catalysis | Rhodobacter capsulatus | urate + NADH + H+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
XDH1 | - |
Rhodobacter capsulatus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Rhodobacter capsulatus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.9 | - |
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 |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Rhodobacter capsulatus |
General Information | Comment | Organism |
---|---|---|
additional information | XDH active site structure with conserved Glu232 and Arg310 residues. Analysis of crystal structure of xanthine dehydrogenase, PDB ID 2W3S, where an ionized glutamate 802/232 acts as a hydrogen bonding acceptor from the substrate N3 nitrogen | Rhodobacter capsulatus |