Chemical modification of tyrosine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: assignment in sequence and catalytic involvement

Abstract

p-Hydroxybenzoate hydroxylase was modified by diethyl pyrocarbonate at pH values greater than 7 and by p-diazobenzoate. Modification of the enzyme by diethyl pyrocarbonate abolishes the affinity of the enzyme for the substrate p-hydroxybenzoate. Modification by p-diazobenzoate has the same effect on the enzyme. The enzyme is protected against these modifications by the effector p-fluorobenzoate. The data indicate that the modification of one tyrosine residue in the active center of the enzyme is responsible for the loss of enzyme activity. This tyrosine residue has been identified by sequence studies using radioactively labeled p-diazobenzoate and was found to be most probably Tyr-222. Diethyl pyrocarbonate reacts with a tyrosine residue in the active center other than Tyr-222; the former could not be identified. Sequence studies further showed that Cys-211 is also partially modified by p-diazobenzoate. In addition, the sequence of residues 343-345 was found to be Ser-Trp-Trp instead of the tentative assignment Ser-Tyr-Trp made earlier. The results are briefly discussed on the basis of the existing three-dimensional model of the enzyme.

Reactivity of 3‐HBA‐6‐Hydroxylase with Diethylpyrocarbonate and Ns‐Bromosuccinimide: Effect of Chemical Modifications on Kinetic and Spectral Properties of the Enzyme

“…Therefore one might conjecture the participation of one or more aromatic residues such as tyrosine and/or tryptophan in catalysis via hydrophobic interactions and hydrogen bonding. Weijer et al (1983) and Wijnands et al (1986) have indeed detected three tyrosine residues at/close to the active center of phydroxybenzoate hydroxylase through chemical modification and protein sequencing studies. They further propose that an ionized tyrosine residue with a pK a value of ∼7.6 is hydrogen bonded to the hydroxyl group of p-HBA to facilitate hydroxylation.…”

“…The dicarbethoxyhistidyl derivative is not dissociated by hydroxylamine (Miles, 1977). Decarbethoxylation of the O-carbethoxytyrosyl residue is a slow process since this complex is 4 times less reactive with hydroxylamine as compared to N-carbethoxyimidazole (Melchoir and Fahrney, 1970;Wijnands et al, 1986).…”

“…Time-dependent increase in the intensity of enzyme emission fluorescence at 335 nm and significant reduction in the molar ellipticity value at 280 nm as seen in the near-UV CD spectrum of the DEPC-inactivated 3-HBA-6-hydroxylase may be indicative of (a) physical interaction of the ligand DEPC with tryptophan/tyrosine residues and/or (b) changes in the microenvironment of tryptophan/tyrosine residues due to DEPC adduction. Wijnands et al (1986) have reported an increase in the fluorescence of the enzyme-bound FAD following DEPC modifications of active site residues Tyr 201 and/or Tyr 385 in p-hydroxybenzoate hydroxylase at pH values exceeding 7.00. However, they did not observe changes in the intrinsic fluorescence properties of the enzyme.…”

“…The binding of a phenolic substrate to oxidized flavoprotein is the most crucial step in the hydroxylation of aromatic compounds by all monooxygenases and has attracted the attention of several investigators. A wealth of information that is available on chemical modifications (Shoun et al, 1980;Shoun and Beppu, 1982;Van Berkel et al, 1984;Wijnands and Muller, 1982;Wijnands et al, 1986;Wijnands et al, 1987), primary structure by sequencing (Hofsteenge et al, 1980;Hofsteenge et al, 1983;Weijer et al, 1982), and tertiary structure by X-ray crystallography Wierenga et al, 1979) and detailed mechanistic studies on p-HBA (p-hydroxybenzoate) hydroxylase has provided in-depth knowledge on the active site residues, the nature of substrate-enzyme interactions, and the chemical basis of ortho hydroxylation, which is frequently encountered with most of the monooxygenases. However, not much progress has been made in understanding the active site of para hydroxylating enzymes such as 3-HBA-6-hydroxylase.…”

Interaction of 3-Hydroxybenzoate with 3-Hydroxybenzoate-6-hydroxylase

“…The substrate binding ability of the NBS-reacted enzyme is half that of the native enzyme. The presence of three tyrosine residues at/close to the active site of p-HBA hydroxylase from Pseudomonas fluorescens was proposed on the basis of chemical modification and protein sequencing data by Weijer et al (1983) and Wijnands et al (1986). These research workers postulated that an ionized tyrosine residue with a pK a value of ∼7.6 is hydrogen bonded to the hydroxyl group of p-HBA to facilitate ring hydroxylation.…”