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Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes.
J Biol Chem. 1989 Jun 25; 264(18):10567-73.JB

Abstract

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.

Authors+Show Affiliations

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

2732238

Citation

Massey, V, et al. "Differences in Protein Structure of Xanthine Dehydrogenase and Xanthine Oxidase Revealed By Reconstitution With Flavin Active Site Probes." The Journal of Biological Chemistry, vol. 264, no. 18, 1989, pp. 10567-73.
Massey V, Schopfer LM, Nishino T, et al. Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes. J Biol Chem. 1989;264(18):10567-73.
Massey, V., Schopfer, L. M., Nishino, T., & Nishino, T. (1989). Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes. The Journal of Biological Chemistry, 264(18), 10567-73.
Massey V, et al. Differences in Protein Structure of Xanthine Dehydrogenase and Xanthine Oxidase Revealed By Reconstitution With Flavin Active Site Probes. J Biol Chem. 1989 Jun 25;264(18):10567-73. PubMed PMID: 2732238.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes. AU - Massey,V, AU - Schopfer,L M, AU - Nishino,T, AU - Nishino,T, PY - 1989/6/25/pubmed PY - 1989/6/25/medline PY - 1989/6/25/entrez SP - 10567 EP - 73 JF - The Journal of biological chemistry JO - J Biol Chem VL - 264 IS - 18 N2 - The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/2732238/Differences_in_protein_structure_of_xanthine_dehydrogenase_and_xanthine_oxidase_revealed_by_reconstitution_with_flavin_active_site_probes_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(18)81658-1 DB - PRIME DP - Unbound Medicine ER -