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Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase.
Biochemistry. 2005 Oct 11; 44(40):13304-14.B

Abstract

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.

Authors+Show Affiliations

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

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16201756

Citation

Frederick, Kendra King, and Bruce A. Palfey. "Kinetics of Proton-linked Flavin Conformational Changes in P-hydroxybenzoate Hydroxylase." Biochemistry, vol. 44, no. 40, 2005, pp. 13304-14.
Frederick KK, Palfey BA. Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase. Biochemistry. 2005;44(40):13304-14.
Frederick, K. K., & Palfey, B. A. (2005). Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase. Biochemistry, 44(40), 13304-14.
Frederick KK, Palfey BA. Kinetics of Proton-linked Flavin Conformational Changes in P-hydroxybenzoate Hydroxylase. Biochemistry. 2005 Oct 11;44(40):13304-14. PubMed PMID: 16201756.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase. AU - Frederick,Kendra King, AU - Palfey,Bruce A, PY - 2005/10/6/pubmed PY - 2006/1/5/medline PY - 2005/10/6/entrez SP - 13304 EP - 14 JF - Biochemistry JO - Biochemistry VL - 44 IS - 40 N2 - p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/16201756/Kinetics_of_proton_linked_flavin_conformational_changes_in_p_hydroxybenzoate_hydroxylase_ L2 - https://doi.org/10.1021/bi051119t DB - PRIME DP - Unbound Medicine ER -