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Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase.
Biochemistry. 2003 Sep 30; 42(38):11234-42.B

Abstract

p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure. In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme. It was discovered that the enzyme with picolinate bound was reduced at a rate similar to that with p-hydroxybenzoate bound at high pH. However, there was a large effect of pH upon the rate of reduction in the presence of picolinate with a pK(a) of 7.4, identical to the pK(a) of picolinate bound to the enzyme. The intensity of charge-transfer bands observed between FAD and NADPH during the reduction process correlated with the rate of flavin reduction. We conclude that high rates of reduction of the enzyme require (a) the isoalloxazine of the flavin be held by the protein in a solvent-exposed position and (b) the movement of a loop of protein so that the pyridine ring of NADPH can move into position to form a complex with the isoalloxazine that is competent for hydride transfer and that is indicated by a strong charge-transfer interaction.

Authors+Show Affiliations

Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.No 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, P.H.S.

Language

eng

PubMed ID

14503873

Citation

Ortiz-Maldonado, Mariliz, et al. "Conformational Changes Combined With Charge-transfer Interactions Are Essential for Reduction in Catalysis By P-hydroxybenzoate Hydroxylase." Biochemistry, vol. 42, no. 38, 2003, pp. 11234-42.
Ortiz-Maldonado M, Entsch B, Ballou DP. Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase. Biochemistry. 2003;42(38):11234-42.
Ortiz-Maldonado, M., Entsch, B., & Ballou, D. P. (2003). Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase. Biochemistry, 42(38), 11234-42.
Ortiz-Maldonado M, Entsch B, Ballou DP. Conformational Changes Combined With Charge-transfer Interactions Are Essential for Reduction in Catalysis By P-hydroxybenzoate Hydroxylase. Biochemistry. 2003 Sep 30;42(38):11234-42. PubMed PMID: 14503873.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase. AU - Ortiz-Maldonado,Mariliz, AU - Entsch,Barrie, AU - Ballou,David P, PY - 2003/9/25/pubmed PY - 2003/10/30/medline PY - 2003/9/25/entrez SP - 11234 EP - 42 JF - Biochemistry JO - Biochemistry VL - 42 IS - 38 N2 - p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure. In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme. It was discovered that the enzyme with picolinate bound was reduced at a rate similar to that with p-hydroxybenzoate bound at high pH. However, there was a large effect of pH upon the rate of reduction in the presence of picolinate with a pK(a) of 7.4, identical to the pK(a) of picolinate bound to the enzyme. The intensity of charge-transfer bands observed between FAD and NADPH during the reduction process correlated with the rate of flavin reduction. We conclude that high rates of reduction of the enzyme require (a) the isoalloxazine of the flavin be held by the protein in a solvent-exposed position and (b) the movement of a loop of protein so that the pyridine ring of NADPH can move into position to form a complex with the isoalloxazine that is competent for hydride transfer and that is indicated by a strong charge-transfer interaction. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/14503873/Conformational_changes_combined_with_charge_transfer_interactions_are_essential_for_reduction_in_catalysis_by_p_hydroxybenzoate_hydroxylase_ L2 - https://doi.org/10.1021/bi030114y DB - PRIME DP - Unbound Medicine ER -