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Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys.
Biochemistry. 1996 Jul 16; 35(28):9278-85.B

Abstract

The isoalloxazine ring system of the FAD cofactor of p-hydroxybenzoate hydroxylase must be secluded from solvent at specific stages of catalysis in order to form and stabilize a flavin C4a-hydroperoxide. This species may then react with the activated phenolate of p-hydroxybenzoate. A number of crystal structures of the enzyme with alterations to active site substituents or complexes with analogue benzoates have revealed an alternate position for the isoalloxazine (Gatti et al. (1994) Science 266, 110-114; Schreuder et al. (1994) Biochemistry 33, 10161-10170). This new flavin conformation is 7 A "out" toward solvent and may open a passage for substrate entry to the active site. Arginine 220 is one of the few residues in the structure to demonstrate conformational changes when the flavin is "out". In this study we have made the Arg220Lys mutant to test the significance of this residue in flavin movement. The R220K mutation has brought about dramatic alterations to all aspects of catalysis. Stopped flow kinetic characterization of the mutant has revealed that, while the effector role for the substrate is maintained, there exists an order of magnitude decrease in the limiting rate of reduction, even though there is 40-fold increase in association with NADPH. The mutant enzyme has only a fraction of its reductive half-reaction coupled to product formation, and the hydroxylation process is slow. This occurs despite a higher proportion of the more activated substrate phenolate in the active site. Many of the observed changes can be attributed to a decrease in the stability of the "in" conformation of the flavin during the catalysis and indicate a role for flavin conformational states in many of the catalytic processes of the enzyme.

Authors+Show Affiliations

Department of Molecular and Cellular Biology, University of New England Armidale, New South Wales, Australia.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, P.H.S.

Language

eng

PubMed ID

8703933

Citation

Moran, G R., et al. "Evidence for Flavin Movement in the Function of P-hydroxybenzoate Hydroxylase From Studies of the Mutant Arg220Lys." Biochemistry, vol. 35, no. 28, 1996, pp. 9278-85.
Moran GR, Entsch B, Palfey BA, et al. Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys. Biochemistry. 1996;35(28):9278-85.
Moran, G. R., Entsch, B., Palfey, B. A., & Ballou, D. P. (1996). Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys. Biochemistry, 35(28), 9278-85.
Moran GR, et al. Evidence for Flavin Movement in the Function of P-hydroxybenzoate Hydroxylase From Studies of the Mutant Arg220Lys. Biochemistry. 1996 Jul 16;35(28):9278-85. PubMed PMID: 8703933.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys. AU - Moran,G R, AU - Entsch,B, AU - Palfey,B A, AU - Ballou,D P, PY - 1996/7/16/pubmed PY - 1996/7/16/medline PY - 1996/7/16/entrez SP - 9278 EP - 85 JF - Biochemistry JO - Biochemistry VL - 35 IS - 28 N2 - The isoalloxazine ring system of the FAD cofactor of p-hydroxybenzoate hydroxylase must be secluded from solvent at specific stages of catalysis in order to form and stabilize a flavin C4a-hydroperoxide. This species may then react with the activated phenolate of p-hydroxybenzoate. A number of crystal structures of the enzyme with alterations to active site substituents or complexes with analogue benzoates have revealed an alternate position for the isoalloxazine (Gatti et al. (1994) Science 266, 110-114; Schreuder et al. (1994) Biochemistry 33, 10161-10170). This new flavin conformation is 7 A "out" toward solvent and may open a passage for substrate entry to the active site. Arginine 220 is one of the few residues in the structure to demonstrate conformational changes when the flavin is "out". In this study we have made the Arg220Lys mutant to test the significance of this residue in flavin movement. The R220K mutation has brought about dramatic alterations to all aspects of catalysis. Stopped flow kinetic characterization of the mutant has revealed that, while the effector role for the substrate is maintained, there exists an order of magnitude decrease in the limiting rate of reduction, even though there is 40-fold increase in association with NADPH. The mutant enzyme has only a fraction of its reductive half-reaction coupled to product formation, and the hydroxylation process is slow. This occurs despite a higher proportion of the more activated substrate phenolate in the active site. Many of the observed changes can be attributed to a decrease in the stability of the "in" conformation of the flavin during the catalysis and indicate a role for flavin conformational states in many of the catalytic processes of the enzyme. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8703933/Evidence_for_flavin_movement_in_the_function_of_p_hydroxybenzoate_hydroxylase_from_studies_of_the_mutant_Arg220Lys_ L2 - https://doi.org/10.1021/bi960360s DB - PRIME DP - Unbound Medicine ER -