A Study on p-Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens

Abstract

Conventional methods to prepare the apoenzyme of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens yield an inhomogeneous apoenzyme with respect to the reconstitutable activity. To overcome this problem the holoenzyme was bound covalently to a Sepharose–5,5′-dithio-bis(2-nitrobenzoate) column via the reactive sulfhydryl group of the enzyme. The prosthetic group FAD was removed at neutral pH by addition of a high concentration of KBr and urea to the elution buffer. The holoenzyme can be reconstituted while the apoenzyme is still bound to the column or the apoenzyme can be isolated in the free state. The yield of apoenzyme was between 90% and 95% of the starting enzyme. The degree of reconstitution of holoenzyme is better than 95% of the original activity. The apoenzyme is stable for a long period of time as an ammonium sulfate precipitate. The relative molecular mass of the apoenzyme is 43000 per polypeptide chain. The apoenzyme exists mainly as a dimer in solution. During the preparation of the apoenzyme the substrate p-hydroxybenzoate protects the apoenzyme from inactivation, indicating complex formation. The dissociation constant for the apoenzyme-substrate complex was determined to be about 30 μM. The apoenzyme also forms a complex with NADPH. The dissociation constant (∼ 12 μM) of this complex is about a factor of ten smaller than that of the holoenzyme (∼ 120 μM). The dissociation constant of the apoenzyme-NADPH complex is strongly dependent on pH and ionic strength of the solution. The dissociation constant of the holoenzyme into its constituents is 45 nM. The association rate constant calculated from kinetic experiments is 4.1 × 104 M−1 s−1. The calculated dissociation rate constant is 1.85 × 10−3 s−1, indicating that the holoenzyme possesses a high stability which is governed by the low dissociation rate constant. The method of preparation of apoenzyme can also be used to separate enzyme molecules where the reactive SH group has been oxidized by air from enzyme molecules still containing the original SH group. This separation technique is in particular useful when quantitative labelling of the SH group is required.