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The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase.
J Biol Chem. 2004 Sep 24; 279(39):40437-44.JB

Abstract

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.

Authors+Show Affiliations

Department of Plant Biology, Technical University Braunschweig, 38023 Braunschweig, Germany. S.Leimkuehler@tu-bs.deNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15265866

Citation

Leimkühler, Silke, et al. "The Role of Active Site Glutamate Residues in Catalysis of Rhodobacter Capsulatus Xanthine Dehydrogenase." The Journal of Biological Chemistry, vol. 279, no. 39, 2004, pp. 40437-44.
Leimkühler S, Stockert AL, Igarashi K, et al. The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase. J Biol Chem. 2004;279(39):40437-44.
Leimkühler, S., Stockert, A. L., Igarashi, K., Nishino, T., & Hille, R. (2004). The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase. The Journal of Biological Chemistry, 279(39), 40437-44.
Leimkühler S, et al. The Role of Active Site Glutamate Residues in Catalysis of Rhodobacter Capsulatus Xanthine Dehydrogenase. J Biol Chem. 2004 Sep 24;279(39):40437-44. PubMed PMID: 15265866.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase. AU - Leimkühler,Silke, AU - Stockert,Amy L, AU - Igarashi,Kiyohiko, AU - Nishino,Takeshi, AU - Hille,Russ, Y1 - 2004/07/20/ PY - 2004/7/22/pubmed PY - 2004/10/27/medline PY - 2004/7/22/entrez SP - 40437 EP - 44 JF - The Journal of biological chemistry JO - J Biol Chem VL - 279 IS - 39 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/15265866/The_role_of_active_site_glutamate_residues_in_catalysis_of_Rhodobacter_capsulatus_xanthine_dehydrogenase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(20)77129-2 DB - PRIME DP - Unbound Medicine ER -