Reactivation of vanadium bromoperoxidase; inhibition by metallofluoric compounds

Bioehimica et BiophysicaActa, 1079(1991153-56 © 1991ElsevierSciencePublishersB.V. (1167-4838/91/$03.50 ADONIS 016748389100252R

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BBAPRO33969

Reactivation of vanadium bromoperoxidase; inhibition by metallofluoric compounds Michiel T r o m p l, T r a n T h a c h V a n 2 a n d R o n W e v e r i J E.C Slater Inslitute for BiocheratcalResearchand BiotechnologicalCentre, Unit'ersityof Amsterdara. Amsterdam (The Netherlands) and 2 Facuhy of Chemistt)', Unh'ersiryof Ha-Noi. Ha-Noi (Vfemam~

(Received5 FebruaryIqgl)

Key words: Bromoperoxidase;Reactivation:Berylliumfluoride;Aluminiumfluoride;~rophosphate;(A. nodomm~ The effect of phosphate analogs (pyrophosphate, aluminofluoride and beryllofluoride complexes) on the reactivation of apohromoperoxidase by vanadate was studied. PzO 4- inhibited the reactivation in the millimolar range. Of the different aluminofluoride complexes, only AIF4- was inhibitory. In addition, BeF42- also appeared to bind with high affinity to the apobromoperoxidase, thus inhibiting the reactivation very strongly. The inhibition observed supports a mechanism in which the fluorometallic complexes act as analogs of vanadate and bind accordingly to the apobromoperoxidase.

Introduction It is known that in the brown seaweed Ascophyllum nodosum two different bromoperoxidases are present, both belonging to the group of haloperoxidases that contain vanadium as an essential element for enzymic activity [1]. These enzymes catalyze the oxidation of bromide by hydrdogen peroxide, resulting in bromination of suitable organic substrates, such as ,8-diketones, ,8-ketoacids, phenols, suifur-heterocycles [21, 2-thiouracil, trans-4-hydroxycinnamic acid [3], barbituric acid and its derivatives [4]. The mechanism of halogenation catalyzed by these bromoperoxidases was studied in detail [4], and it was shown that for these enzymes, HOBr is probably the primary enzym;c product. A general property of these enzymes is that the vanadium can be removed from bromoperoxidase by dialysis at low pH, in the presence of EDTA and phosphate, resulting in an inactive apo-enzyme [5,6]. The enzymic activity can be fully restored by addition of orthovanadate (VO 3-) to the ~,po-enzyme [7]. The reactivation process is inhibited by structural analogs of vanadate, such as phosphate (PO~-), molybdate (MoO~-)[7-91 and arsenate (AsO.~-) [10]. According to these studies, the inhibition caused by these species is due to compe-

Correspondence:MG.M Tromp. E.C. SlaterInstitutefor Biochemical Researchand BiolechnologicalCenler, Universityof Amsterdam, Plant~geMuidergracht12. 1018TV Amsterdam.The Netherlands.

tition with vanadate for the active site of the enzyme. In contrast, Everett et ai. [11] suppose that inhibition by phosphate is due to a reaction with vanadate, leading to the formation of a mixed poly-oxo species. Sternweis and Gilman [t2] made the key observation that activation of G-protein by fluoride was dependent on the presence of AI 3÷. Since then, it has become clear that the fluoroaluminates were in fact responsible for the observed activation. Fluoride and aluminium ions are in equilibrium to form aluminofluoride complexes (AIF~3- ~), and the nature of the actual complex depends on the concentration of fluoride present in .solution [14]. Recently it was again stressed by Chabre [13] that aluminofluoride and beryllofluoride complexes can act as phosphate analogs in enzymology. He stated that these complexes, rather than the direct action of fluoride, inhibited enzymes like phosphatases or ATPases. This prompted us to study the t:ifect of these complexes and the pyrophosphate anion (PzO~-) on the reactivation of bromoperoxidase by vanadate. Materials and Methods Bromoperoxidase was isolated from Ascophyllum nodosum as described in Ref. 8. Apobromoperoxidase was prepared by dialysis of the enzyme against a 0.1 M citrate/phosphate buffer with 1 mM EDTA, as reported by Viiter [5] and De Boer et al. [61. For the reconstitution studies in the presence of P20-~-, aluminofluoride or the beryllofluoride complexes, the apo-

54 bromoperoxidasc was incubated with thcsc species for 24 h at room temperature in 0.1 M Tris-SO4 (pH 8.3), 0.2 M Na,SO~, in the presence of an appropriate amount of VOW-. Enzymic activity of bromoperoxidasc was mcasurcd by following the bromination of monochlorodimedone spectrophotomctrically at 290 nm. The standard assay mixturc contained 2.5 ml of (1.I M potassium phosphate (pH 6.5), (}.2 M N a , S Q , 0.1 M KBr and 50/,tM monochlorodimcdonc. After addition of 100 p.l of incubated cnD'mc, the reaction was started by adding 2 mM H20,. Optical spectroscopy was performed on a Cary-17 spectrophotometer. The vanadate, pyrophosphate, fluoride, aluminium and beryllium solutions were prepared by dissolving Na~VOa, NaaP2Or, KF, A12(SO4)~ and BeSO~ in water. Water was purified, using an Elgastad B124 (Elga group) and a Milli-O (Millipore corporation) water purification unit. Prior to use, all buffers were extensively dialyzed against Chelex-100 cation-exchanging resin (Bio-Rad), to remove trace amounts of metal ions. (Especially A13+ is known to be a frequent contaminant of laboratory glassware.) Na4P20 7 was purchased from Sigma. All chemicals used were of analytical grade. Results

Effecls of aluminofluoride and beryllofluoridecomplexex The fluoride complexes of aluminium or beryllium have been recognized as a new class of phosphate analogs [13]. Since phosphate inhibited the reactivation of apobromoperoxidase [7-10], the effect of these complexes on the reactivation of the apobromoperoxidase was investigated, using various concentrations of F-, AI3+ and Be TM ions. Fig. I shows the rcconstitution of apo-enzymc by vanadate, as measured by the enzymic activity, as a function of both the fluoride and aluminium concentrations. It is apparent that high con-

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i.o~1 ~ ' l l j | Fig. I. Relative activity of bromoperoxidase after rcconstitution by vanadate, as a function of both the aluminiurn concentration ([AI 3' ]) and the fluoride concentration (IF- ]). Tl:c concentration of apobromoperoxida~ was 30 nM.

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Fig. 2. Activityof bromoperoxidaseafter reconstitutionby vanadate. in the presence of different concentrationsof beryllium([Be:* ]). The fluorideconcentration'wasset at 50 raM.

centrations of either one of these species leads to inhibition of the reconstitution. However, in combination, especially at low aluminium concentrations and at fluoride concentrations of 5 to 10 raM, the inhibition of the reactivation is most pronounced, From equilibrium constants of metal fluoride complexes [14] it is known that at these concentrations of F- the predominant species present is AIFf. Further increasing the fluoride concentration to 50 mM leads to a decrease in inhibition, which is very likely due to the formation multifluorinated species, such as AIFsz-, which do not resemble phosphate (or vanadate'l and therefore are not inhibitory. This also strongly indicates that the inhibition at lower fluoride concentrations is reversible. A further increase of fluoride, in ti;e presence of a fixed concentration of AI 3. (0.02-2 raM) again shows an increase in inhibition. This may be related to the observation that fluoride alone, in the absence of aluminium is already inhibitory. A decrease in the inhibition is observed when, at a fixed fluoride concentration of 5 raM, the aluminium concentration is raised above 2 raM. This may be due to formation of complexes with lower fluoride content (e.g., AIF3, or AIF2+). Here also, the analysis is hampered by the fact that high concentrations of aluminium, in the absence of fluoride, are already inhibitory. At high concentrations of both aluminium (4-8 mM) and fluoride (100 mM), far less inhibition is observed. This is very likely due to the formation of non-inhibitory complexes. Beryllofluoride complexes have a similar, though less complex effect on the reconstitution as aluminoflupride complexes. Addition of beryllium at concentrations of fluoride of 50 to 100 mM leads to the formation BeF~- (higher fluorinated beryllium complexes than BeF~- are not formed) and, as can be seen from Fig. 2, these beryllofluoride complexes are highly inhibitory to the reactivation, with a K. of at about 2 gM.

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PYROPHOSPHATE ImM) Fig. 3. Dixon plot of the inhibition by pyrophosphate of the reconstitulion O[ bromoperoxidase. The apo-enz~me concentration was 25 riM.; o o. 50 nM VOW-" • • 151) mM VO~ . The inhibition constant tK,) ~as delermined according to the mctht~d described by Dixon [t5].

Pyrophosphate #zhibition Fig. 3 shows that pyrophosphate is inhibitory in the millimolar range (K i = 50 raM) and that the inhibition is competitive with respect to vanadate [15]. In comparison to phosphate inhibition [10], the inhibition is about 1000-times tess strong. It should be noted that this apparent inhibition may be due to traces of phosphate in the pyrophosphate solution. Discussion The inhibition of the reconstitution, caused by aluminium fluoride shows that maximal inhibition occurs at a fluoridt, concentration of 5 to I0 mM At these concentrations the predominant aluminofluoride complex is AIF4- [14]. This species is a very strong inhibitor, since very low concentrations of aluminium are already inhibitory. In buffer systems which were not treated with Chelex-100, inhibition was already observed without addition of aluminium. The amount of aluminium, contaminating water and buffers stored in laboratory glassware is estimated in the order of micromolars [13]. That the inhibition observed is not due to fluoride solely, but to aluminofluoride complexes is supported by the observation that in the presence of moderate amounts of aluminium [20-100 p.M). the reactivation by vanadate is less inhibited at high fluoride concentrations. We estimate the inhibition constant of the AIF~- species in the order of a few micromolars. The reasons why either aluminium or fluoride solely are inhibitory at high concentrations are not clear. Addition of inhibitory amounts of aluminiium or fluoride to an assay of holo-enzy-me did not affect the brominating activity. In addition, prolonged preincubation of the holo-enzyme (30 nM} in 4 mM A1J~ or in 100 mM F- had no effect on the enzymic activity.

Like aluminium, beryllofluoride complexes clearly show inhibition (Fig. 2). At fluoride concentrations of 5(1 to 100 mM the predominant beryllofluoridc species is Bc[~2 . The inhibition by this sp,~cics is ~!r,'.'~dy obscr¢cd at vcr"s low concentrations, demonstrating that that the affinity for the apo-enzyme is very high. Everett ctal. [11] suggested that the inhibition of the reactivation caused by phosphate was due to the formation of mixed poly-anions with vanadate, thus reducing the amount of free vanadate that can bind to the apo-enzyme. The ability of either phosphate or pyrophosphate to form complexes with vanadate, is of the same order of magnitude [16], so the inhibition of the reactivation caused by either of these species should be in the same order of magnitude. This is clearly not the case, the inhibition observed in the presence of phosphate ( K , = ~ ) ~M) [10] is lO00-times stronger than the inhibition caused by pyrophosphate. Therefore we conclude that the inhibition observed is not due to the formation of mixed poly-anions. Rather these studies are in line with our original proposal in which phospl~ale and phosphate analogs (which resemble vanadate) occupy the vacant vanadate binding site in the apo-enzyme. '~'ever and Kustin [17] suggested that it is very likely that upon reactivation of the apo-enzyme by vanadate, the vanadium-oxs'gen bonds are retained. The vanadate will thus be incorporated as a tetrahedral-like compound in the apo-enzyme, and will possibly bc held in place by two additional ligand donors coming from the protein moiety.. This sugge~ tion is supported by the finding that a strictly tetrahedrai compound, such as berrylofluoride ( B e F ~ ) [t3] is able to inhibit the reincorporafion of vanadate so effectively. Acknowledgements One of us (Tran Thach Van) wishes to thank the University of Amsterdam for a grant, cTlabling him to stay and to carry, out this research within the framework of the project VH I. We thank Dr. R. de Vries for realization of this cooperation. Mr. M.B.L Rientjes is acknowledged for his help constructing Fig. 1. This work is part of the research program of the Netherlands Foundation for Chemicai Research (SON.). References 1 Krenn. B.E.. Tromp. M.GM. and Wever. R. (1989} J. Biol. Chem. 2ca, 1q287-19292. 2 Neidleman~ S.L, and Geigert (lqgh) Biohalogenation: Principles, Basic Roles and Applications, Ellis Horg'ood Ltd.. Chiehester 3 De Boer, E and Wever, R (1988) J. Biol, Chem. 263. 12326 12332 4 Ftaas~n, M.CR.. Jansma, J D.. Van der Pins. tt.C, De Boer, E. and Weber, R (198,'0 Bioorg. Chem. 16. 352-363. 5 Viller. H. (1984) Ph~Lx~hem. 23, 1387-1390.

56 6 De Boer, E.. Tromp, M.GM., Plat, I1., Krenn, G.E, and Wever, R. 11986) Biochim Biophys. Acta 872, I(H-115. 7 De Boer, E., Boon, K. and Wever, R, fI988) Biochemistry 27, 1629-1635. 8 De Boer, E.. Van Kooyk, Y., Tromp, M.G.M., Plat. H. and Wever, R. 11086) Biochim. Biophys. Acta 872, 104-115. 0 Wever, R. Krenn. B.E. De Boer, E., Offenberg, H. and Plat, H. 11988) Oxidases and Related Redox Systems, pp. 477-.493, Alan R. Liss. New York. 10 Tromp, MG.M., Olafsson, G., Krenn, B.E and Wever. R. (19~)1 Bioehim. Biophys, Aeta 1040. 192-198.

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