Chemical modification of sulfhydryl groups in p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens

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Abstract
The cysteine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens were modified with several cysteine reagents. One of the five sulfhydryl groups reacts rapidly and specifically with N-ethylmaleimide without inactivation of the enzyme. Cysteine-116 was found to be the reactive cysteine by isolation of a labeled tryptic peptide. The enzyme is easily inactivated by mercurial compounds. The original activity can be fully restored by treatment of the modified enzyme with sulfhydryl-containing compounds. The rate of incorporation of mercurial compounds is pH-independent and is pseudo-first-order up to 90-95% loss of activity. The reaction shows saturation kinetics. The substrate p-hydroxybenzoate protects the enzyme from fast inactivation. The mercurial compounds themselves inhibit the inactivation reaction at concentrations higher than 80 microM. A spin-labeled derivative of p-chloromercuribenzoate reacts fairly specifically with only Cys-152 on use of enzyme prelabeled with N-ethylmaleimide, in contrast to p-chloromercuribenzoate which reacts with additional cysteine residues, i.e. Cys-211 and Cys-158. From these results it is concluded that modification of Cys-152 decreases drastically the affinity of the enzyme for the substrate. The results strongly indicate that the substrate binding site and Cys-152 are interdependent. This observation is not obvious when the three-dimensional data only are considered. The modified enzyme exhibits a somewhat higher affinity for NADPH than the native enzyme. Modification of N-ethylmaleimide-prelabeled enzyme by p-chloromercuribenzoate leads to absorbance difference spectra showing maxima at 250 nm, 290 nm and 360 nm. The intensities of the absorbance difference maxima at 290 nm and 360 nm are strongly dependent on the pH value of the solution. The intensities are very low at low pH values and increase with increasing pH values, reaching a maximum at about pH = 9. The ionizing group shows a pK value of about 7.6. The maximal molar difference absorption coefficient at 290 nm is 3200 M-1cm-1 at pH 9, suggesting that tyrosine residues ionize under the conditions of modification of the enzyme. The results are discussed in the light of the known three-dimensional structure.