| Abstract
| - p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-componentflavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzymeby NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reducedFAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts withp-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated throughconformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently,it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structureof a mutant form of the enzyme without substrate was published that showed an open conformation withsolvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., andGatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608−613]. The wild-type enzyme does not formhigh-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substratealso forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea,we have studied the properties of two different mutant forms of the enzyme that are stabilized in the openconformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, asexpected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowlyforming small amounts of product by the normal catalytic pathway. The lack of activity results from thefailure of the mutants to readily form the out conformation required for flavin reduction by NADPH. Themutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoatebound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formationof this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. Thisoverall study emphasizes the role that protein dynamics can play in enzymatic catalysis.
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