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Soybean nodule xanthine dehydrogenase: a kinetic study.
Arch Biochem Biophys. 1983 Apr 15; 222(2):435-41.AB

Abstract

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.

Authors

No 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, Non-P.H.S.

Language

eng

PubMed ID

6573870

Citation

Boland, M J., et al. "Soybean Nodule Xanthine Dehydrogenase: a Kinetic Study." Archives of Biochemistry and Biophysics, vol. 222, no. 2, 1983, pp. 435-41.
Boland MJ, Blevins DG, Randall DD. Soybean nodule xanthine dehydrogenase: a kinetic study. Arch Biochem Biophys. 1983;222(2):435-41.
Boland, M. J., Blevins, D. G., & Randall, D. D. (1983). Soybean nodule xanthine dehydrogenase: a kinetic study. Archives of Biochemistry and Biophysics, 222(2), 435-41.
Boland MJ, Blevins DG, Randall DD. Soybean Nodule Xanthine Dehydrogenase: a Kinetic Study. Arch Biochem Biophys. 1983 Apr 15;222(2):435-41. PubMed PMID: 6573870.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Soybean nodule xanthine dehydrogenase: a kinetic study. AU - Boland,M J, AU - Blevins,D G, AU - Randall,D D, PY - 1983/4/15/pubmed PY - 1983/4/15/medline PY - 1983/4/15/entrez SP - 435 EP - 41 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 222 IS - 2 N2 - Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/6573870/Soybean_nodule_xanthine_dehydrogenase:_a_kinetic_study_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0003-9861(83)90542-8 DB - PRIME DP - Unbound Medicine ER -