The importance of monopole‐monopole and monopole‐dipole interactions on the binding of NADPH and NADPH analogues to p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens

Abstract

NADPH binding to p-hydroxybenzoate hydroxylase from Pseudornonas JZuorescens is found to be strongly dependent on pH and ionic strength. In the ionic strength range of 0.02-0.15 M, optimal NADPH binding is observed at a pH value of 6.4. Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values > 8. Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate.The experimental data can be explained by a theoretical model in which monopole-monopole or monopoledipole interactions between the enzyme and the ligand are dominant. Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation. The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5. The results are discussed with respect to published results, including data obtained from the enzyme from a different source.p-Hydroxybenzoate hydroxylase from Pseudornonas jluorescens is an external flavoprotein monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to 3,4-dihydroxybenzoate. The reaction requires NADPH as an electron donor [l]. It has been observed that the reduction of the proteinbound FAD by NADPH is accelerated enormously upon substrate binding [2]. This 'effector' role was shown to be a property of several substrate analogues, which are not necessarily transformed to a product [3]. The enhancement of the rate of reduction of FAD cannot be explained by an increase in NADPH binding since the dissociation constant of the enzyme-NADPH complex decreases only slightly at pH 6.6 and about 10-fold at pH 7.5 in the presence of the substrate or effector [I]. It has been suggested, for the enzyme from Pseudornonas desrnolytica, that the substrate p-hydroxybenzoate not only stimulates the rate of reduction of FAD by NADPH but also shifts the pH optimum of the binding of NADPH to more alkaline pH values [4]. This proposal is related to the fact that optimum binding of NADPH or NADH to the free enzyme occurs at about pH 6 whereas the enzyme possesses its optimal activity at a pH value of about 8. The data of the apparent pH-dependent dissociation constants for the complex between enzyme and NADPH or NADH Abbreviations. t-NADPH, 1,4,5,6-tetrahydronicotinamide adenine dinucleotide 2'-phosphate; Mes, 4-morpholineethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid ; Hepps, 4-(2-hydroxyethyl)-l -piperazinepropanesulfonic acid ; 2',5'-ADP, adenosine 2',5'-bisphosphate.Enzyme. p-Hydroxybenzoate hydroxylase (EC 1.14.1 3.2). Definition. Debye, unit of dipole moment = 3.34 x 10-30 ml.served as further support for this proposal. They parallel those of the apparent pH-dependent rat...

Chemical modification of arginine residues in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens: a kinetic and fluorescence study

“…In addition the dissociation constants of the complex between native enzyme and NADPH, and butanedione-modified enzyme and NADPH are very simular, i.e. 130 pM [3] and 165 pM respectively, at pH 6.5 and I = 25 mM (20°C). On the other hand, the corresponding dissociation constants of the p-hydroxybenzoate-enzyme complexes are 35 pM and 150 pM, respectively.…”

“…Fresh solutions of the enzyme in the appropriate buffer were prepared daily by gel filtration over Bio-Gel P-6DG. The buffers were brought to a constant ionic strength of 25 mM with Na2S04 whenever NADPH was present in the incubation mixtures [3]. The enzyme concentration was determined spectrophotometrically on the basis of the FAD content by the use of the molar absorption coefficient of 11.3 mM-' cm-' at 450 nm [16].…”

“…The role of the flavin in the catalytic mechanism, as revealed mainly by kinetic and spectroscopic analyses, has had much attention [l, 21. As far as the role of amino acids residues in the reaction mechanism is concerned studies on the pH dependence of binding of NADPH to the protein [3] and chemical modification [4-91 have given interesting results, especially when related to the known primary and tertiary structure [lo-121.The amino acids residues that have been modified so far include cysteine [9], histidine [5,7,8], arginine [6] and tyrosine [13]. The modification of these residues leads to loss of activity in all four cases.…”

4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3

“…pH-dependent activity measurements were performed in 80 mM Mes, pH 5-7, 80 mM Hepes, pH 7-8, 80 mM Hepps, pH 7.5-8.5, and 80 mM Ches, pH 8.5-9.5. The ionic strength of the Good buffers was adjusted to 100 mM with added sodium sulfate (Wijnands et al, 1984). The inhibition by monovalent anions was studied, essentially as described before (Steennis et al, 1973).…”

Rapid relaxation processes in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens revealed by subnanosecond-resolved laser-induced fluorescence

“…Stock solutions of the enzyme were prepared in the appropriate buffers by gel filtration over Bio-Gel P-6DG. Enzyme solutions of different pH values were obtained by dilution of an aliquot of a stock solution into Mes (pH < 7), Hepes (7 < pH < 8) or Hepps (pH > 8) buffer with the desired ionic strength as described by Wijnands et al [9]. The buffers were brought to the desired pH values and ionic strengths by addition of a NaOH and Na,SO, solution, respectively [9].…”