The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase.
Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases. Here we first describe reductive titration and steady state kinetics on recombinant wild-type R. capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730. The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein. No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant. This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis.
Leimkühler
Silke
S
Department of Plant Biology, Technical University Braunschweig, 38023 Braunschweig, Germany. S.Leimkuehler@tu-bs.de
Stockert
Amy L
AL
Igarashi
Kiyohiko
K
Nishino
Takeshi
T
Hille
Russ
R
D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004227:Dithionite / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009243:NAD / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013455:Sulfur / Q000737:chemistry; D013997:Time Factors; D014466:Ultraviolet Rays; D014527:Uric Acid / Q000737:chemistry; D019820:Xanthine / Q000737:chemistry; D014968:Xanthine Dehydrogenase / Q000737:chemistry*
D009243:NAD; D004227:Dithionite; D019820:Xanthine; D014527:Uric Acid; D018698:Glutamic Acid; D013455:Sulfur; D014968:Xanthine Dehydrogenase; D010100:Oxygen