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1.
The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox).  相似文献   

2.
Cole LJ  Gatti DL  Entsch B  Ballou DP 《Biochemistry》2005,44(22):8047-8058
p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis.  相似文献   

3.
Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.  相似文献   

4.
Frederick KK  Palfey BA 《Biochemistry》2005,44(40):13304-13314
p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.  相似文献   

5.
Zheng Y  Dong J  Palfey BA  Carey PR 《Biochemistry》1999,38(51):16727-16732
X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those for flavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out". There are several spectral regions where characteristic differences exist for flavin in the "in" or "out" conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-)(1). These spectral features can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly "out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).  相似文献   

6.
Proline 293 of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa is in a highly conserved region of the flavoprotein aromatic hydroxylases. It is thought to impart rigidity to the backbone, as it partially cradles the FAD in these hydroxylases. Thus, this residue has been substituted with serine by site-directed mutagenesis to investigate the importance of flexibility of the peptide segment in catalysis. Differential scanning calorimetry demonstrated that the mutation has decreased the stability of the folded mutant protein compared to the wild-type PHBH. The increased flexibility in the protein backbone enhanced the accessibility of the flavin hydroperoxide intermediate to the solvent, causing an increase in the elimination of H(2)O(2) from this labile intermediate and, consequently, a decrease in the efficiency of substrate hydroxylation. Additionally, the increased accessibility of this mutant form of the enzyme makes it more susceptible than the wild-type enzyme to being trapped in the hydroxyflavin intermediate form in the presence of high levels of p-hydroxybenzoate. The mutation also lowers the pK(a) of the phenolic oxygen of bound p-hydroxybenzoate, and eliminates the pH dependence of the rate constant for flavin reduction by NADPH. These experimental observations lead to a model that explains how the wild-type protein can sense the charge of the 4-substituent of the aromatic ligand and link this charge to a flavin conformational change that is required for reaction with NADPH: (i) The peptide oxygen of Pro 293 is repelled by the negative charge of the phenolic oxygen of p-hydroxybenzoate. (ii) This repulsion is transmitted through the peptide backbone, causing the movement of Asn 300. (iii) The change in the position of Asn 300 triggers the movement of the flavin from the largely buried "in" conformation to the exposed, reactive "out" conformation.  相似文献   

7.
The oxygen transfer to p-hydroxybenzoate catalyzed by p-hydroxybenzoate hydroxylase (PHBH) has been shown to occur via a C4a-hydroperoxide of the flavin. Two factors are likely to be important in facilitating the transfer of oxygen from the C4a-hydroperoxide to the substrate. (a) The positive electrostatic potential of the active site partially stabilizes the negative charge centered on the oxygen of the flavin-C4a-alkoxide leaving group during the transition state [Ortiz-Maldonado, M., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 8124-8137]. (b) The hydrogen-bonding network ionizes the substrate to promote its nucleophilic attack on the electrophilic C4a-hydroperoxide intermediate [Entsch, B., Palfey, B. A., Ballou, D. P., and Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. This ionization is also aided by the positive electrostatic potential of the active site [Moran, G. R., Entsch, B., Palfey, B. A., and Ballou, D. P. (1997) Biochemistry 36, 7548-7556]. Substituents on the flavin can specifically affect the stability of the alkoxide leaving-group, whereas changes to specific enzyme residues can affect the charge in the active site and the hydrogen-bonding network. We have used wild-type (WT) PHBH and several mutant forms, all with normal FAD and with 8-Cl-FAD substituted for FAD, to assess the relative contributions of the two effects. Lys297Met and Asn300Asp have decreased positive charge in the active site, and these variants engender approximately 35-fold slower hydroxylation rates than the WT enzyme. Substitution of 8-Cl-FAD in these mutant forms gives approximately 1.8-fold increases in hydroxylation rates, compared with a > or =4.8-fold increase for WT with this flavin. The hydroxylation catalyzed by Tyr385Phe, a mutant enzyme form with a disrupted hydrogen-bonding network that compromises the ionization of the substrate without changing the positive charge of the active site, is stimulated 1.5-fold by substituting the enzyme with 8-Cl-FAD. The substrate, tetrafluoro-p-hydroxybenzoate, is fully ionized in WT PHBH, but this phenolate is a poor nucleophile because of the electron-withdrawing effects of the fluorine substituents. With tetrafluoro-p-hydroxybenzoate as the substrate, substitution of FAD with 8-Cl-FAD in the WT enzyme stabilizes the leaving alkoxide and leads to a 2.3-fold increase in the hydroxylation rate compared to that with FAD. Either the use of substrates that do not communicate with the proton network or the mutation of amino acid residues that perturb this interaction may prevent a necessary conformational change that allows proper orientation between reactants during the hydroxylation reaction or permits the essential protonation of the initially formed nascent flavin-C4a-peroxide anion. Thus, both activation of substrate by the proton network and stabilization of the leaving alkoxide appear to be important for oxygen transfer catalyzed by PHBH. The full effect of the substituents on the flavin (4.8-fold) can only be realized when the optimal transition state can be achieved, and this optimal state is not fully realized with the mutant forms.  相似文献   

8.
Meneely KM  Lamb AL 《Biochemistry》2007,46(42):11930-11937
Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism.  相似文献   

9.
DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.  相似文献   

10.
Frederick KK  Ballou DP  Palfey BA 《Biochemistry》2001,40(13):3891-3899
p-Hydroxybenzoate hydroxylase (PHBH) hydroxylates activated benzoates using NADPH as a reductant and O(2) as an oxygenating substrate. Because the flavin, when reduced, will quickly react with oxygen in either the presence or absence of a phenolic substrate, it is important to regulate flavin reduction to prevent the uncontrolled reaction of NADPH and oxygen to form H(2)O(2). Reduction is controlled by the protonation state of the aromatic substrate p-hydroxybenzoate (pOHB), which when ionized to the phenolate facilitates the movement of flavin between two conformations, termed "in" and "out". When the hydrogen bond network that provides communication between the substrate and solvent is disrupted by changing its terminal residue, His72, to Asn, protons from solution no longer equilibrate rapidly with pOHB bound to the active site [Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158]. Thus, one population of the His72Asn enzyme reduces rapidly and has the phenolate form of pOHB bound at the active site and the flavin in the out conformation. The remaining population of the His72Asn enzyme reduces slowly and has the phenolic form of pOHB bound and the flavin in the in conformation. We have investigated the mechanisms of proton transfer between solvent and pOHB bound to the His72Asn form of the enzyme by double-mixing and single-mixing stopped-flow experiments. We find that, depending on the initial ionization state of bound pOHB and the new pH of the solution, the ionization/protonation of pOHB proceeds through the direct reaction of hydronium or hydroxide with the enzyme-ligand complex and leads to the conversion of one flavin conformation to the other. Our kinetic data indicate that the enzyme with the flavin in the in conformation reacts in two steps. Inspection of crystal structures suggests that the hydroxide ion would react at the re-face of the flavin, and its reaction with pOHB is limited by the movement of Pro293, a conserved residue in similar flavoprotein hydroxylases. We hypothesize that this type of breathing mode by the protein may have been used to compensate for the lack of an efficient proton-transfer network in ancestral hydroxylases, permitting useful catalysis prior to the emergence of specialized proton-transfer mechanisms.  相似文献   

11.
p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.  相似文献   

12.
Apo-p-hydroxybenzoate hydroxylase was reconstituted using 2'-fluoro-2'-deoxy-arabino-FAD, a synthetic flavin in which the hydroxyl of the 2'-center of the ribityl chain was replaced with fluorine in an inverted configuration. The absorbance spectral changes caused by the binding of either p-hydroxybenzoate (pOHB) or 2,4-dihydroxybenzoate (2,4-diOHB) indicated that the isoalloxazine of the artificial flavin adopts the more solvent-exposed "out" conformation rather than the partially buried "in" conformation near the aromatic substrate. In contrast, the flavin of the natural enzyme adopts the in conformation when pOHB is bound. Much of the behavior of the artificial enzyme can be rationalized in light of the preference of the flavin for the out conformation, including the weaker binding of pOHB, the tighter binding of 2,4-diOHB, and the slower reactions involved in the hydroxylation of pOHB and 2,4-diOHB. Particularly noteworthy is the enhancement of the reduction of the flavin by NADPH when pOHB is bound to the active site, consistent with the recent finding that the reaction occurs when the flavin adopts the out conformation (Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158). Thus, whereas the change that induces the out conformation is detrimental to the oxidative half-reaction, it improves the reductive half-reaction, showing that the control of the flavin position in p-hydroxybenzoate hydroxylase represents a compromise between the conflicting needs of two chemically disparate half-reactions, and demonstrating that the 2'-hydroxyl of FAD can serve as a critical control element in flavoenzyme catalysis.  相似文献   

13.
The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens, was replaced by 6-hydroxy-FAD (an extra hydroxyl group on the carbon at position 6 of the isoalloxazine ring of FAD). The catalytic cycle of this modified enzyme was analyzed and compared to the function of native (FAD) enzyme. Transient state kinetic analyses of the multiple changes in the chemical state of the flavin were the principal methods used to probe the mechanism. Four known substrates of the native enzyme were used to probe the reaction. With the natural substrate, p-hydroxybenzoate, the 6-hydroxy-FAD enzyme activity was 12-15% of native enzyme, due to a slower release of product from the enzyme, and less than one product molecule was formed per NADPH oxidized, due to an increased rate of nonproductive decomposition of the transient peroxyflavin essential to the catalytic pathway. More extensive changes in mechanism were observed with the substrates, 2,4-dihydroxybenzoate and p-aminobenzoate. The results suggest that, during catalysis, when the reduced state of FAD is ready for oxygen reaction, the substrate is located below and close to the C-4a/N-5 edge of the isoalloxazine ring. The nature of the high extinction, transient state of flavin, formed upon transfer of oxygen to substrate is discussed. It is not a flavin cation, and is unlikely to be an oxygen-substituted analogue of N-3/C-4 dihydroflavin.  相似文献   

14.
p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.  相似文献   

15.
Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD   总被引:2,自引:0,他引:2  
The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.  相似文献   

16.
para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyses a reaction in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in response to binding p-hydroxybenzoate to the enzyme and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the protein and isoalloxazine ring during catalysis. Earlier research showed that reduction of FAD occurs when the isoalloxazine of the FAD moves to the surface of the protein to allow hydride transfer from NADPH. This move is coordinated with protein rearrangements that are triggered by deprotonation of buried p-hydroxybenzoate through a H-bond network that leads to the surface of the protein. In this paper, we examine the involvement of this same H-bond network in the oxygen reactions-the initial formation of a flavin-C4a-hydroperoxide from the reaction between oxygen and reduced flavin, the electrophilic attack of the hydroperoxide upon the substrate to form product, and the elimination of water from the flavin-C4a-hydroxide to form oxidized enzyme in association with product release. These reactions were measured through absorbance and fluorescence changes in the FAD during the reactions. Results were collected over a range of pH for the reactions of wild-type enzyme and a series of mutant enzymes with the natural substrate and substrate analogues. We discovered that the rate of formation of the flavin hydroperoxide is not influenced by pH change, which indicates that the proton required for this reaction does not come from the H-bond network. The rate of the hydroxylation reaction increases with pH in a manner consistent with a pK(a) of 7.1. We conclude that the H-bond network abstracts the phenolic proton from p-hydroxybenzoate in the transition state of oxygen transfer. The rate of formation of oxidized enzyme increases with pH in a manner consistent with a pK(a) of 7.1, indicating the involvement of the H-bond network. We conclude that product deprotonation enhances the rate of a specific conformational change required for both product release and the elimination of water from C4a-OH-FAD.  相似文献   

17.
Abe I  Kashiwagi K  Noguchi H 《FEBS letters》2000,483(2-3):131-134
Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.  相似文献   

18.
p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational 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 structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also 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 open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis.  相似文献   

19.
The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.  相似文献   

20.
2-Hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44) from Pseudomonas azelaica HBP1 is an FAD-dependent aromatic hydroxylase that catalyzes the conversion of 2-hydroxybiphenyl to 2, 3-dihydroxybiphenyl in the presence of NADH and oxygen. The catalytic mechanism of this three-substrate reaction was investigated at 7 degrees C by stopped-flow absorption spectroscopy. Various individual steps associated with catalysis were readily observed at pH 7.5, the optimum pH for enzyme turnover. Anaerobic reduction of the free enzyme by NADH is a biphasic process, most likely reflecting the presence of two distinct enzyme forms. Binding of 2-hydroxybiphenyl stimulated the rate of enzyme reduction by NADH by 2 orders of magnitude. The anaerobic reduction of the enzyme-substrate complex involved the formation of a transient charge-transfer complex between the reduced flavin and NAD(+). A similar transient intermediate was formed when the enzyme was complexed with the substrate analog 2-sec-butylphenol or with the non-substrate effector 2,3-dihydroxybiphenyl. Excess NAD(+) strongly stabilized the charge-transfer complexes but did not give rise to the appearance of any intermediate during the reduction of uncomplexed enzyme. Free reduced 2-hydroxybiphenyl 3-monooxygenase reacted rapidly with oxygen to form oxidized enzyme with no appearance of intermediates during this reaction. In the presence of 2-hydroxybiphenyl, two consecutive spectral intermediates were observed which were assigned to the flavin C(4a)-hydroperoxide and the flavin C(4a)-hydroxide, respectively. No oxygenated flavin intermediates were observed when the enzyme was in complex with 2, 3-dihydroxybiphenyl. Monovalent anions retarded the dehydration of the flavin C(4a)-hydroxide without stabilization of additional intermediates. The kinetic data for 2-hydroxybiphenyl 3-monooxygenase are consistent with a ternary complex mechanism in which the aromatic substrate has strict control in both the reductive and oxidative half-reaction in a way that reactions leading to substrate hydroxylation are favored over those leading to the futile formation of hydrogen peroxide. NAD(+) release from the reduced enzyme-substrate complex is the slowest step in catalysis.  相似文献   

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