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Metal-Ion Mutagenesis

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Metal-Ion Mutagenesis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by MURAL - Maynooth University Research Archive Library Published on Web 05/26/2009 Metal-Ion Mutagenesis: Conversion of a Purple Acid Phosphatase from Sweet Potato to a Neutral Phosphatase with the Formation of an Unprecedented Catalytically Competent MnIIMnII Active Site Natasˇa Mitic´,† Christopher J. Noble,‡ Lawrence R. Gahan,† Graeme R. Hanson,*,‡ and Gerhard Schenk*,† School of Chemistry and Molecular Biosciences, and Centre of Magnetic Resonance, The UniVersity of Queensland, Queensland, Australia, 4072 Received February 1, 2009; E-mail: [email protected]; [email protected] Abstract: The currently accepted paradigm is that the purple acid phosphatases (PAPs) require a heterovalent, dinuclear metal-ion center for catalysis. It is believed that this is an essential feature for these enzymes in order for them to operate under acidic conditions. A PAP from sweet potato is unusual in that it appears to have a specific requirement for manganese, forming a unique FeIII-µ-(O)-MnII center under catalytically optimal conditions (Schenk et al. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 273). Herein, we demonstrate, with detailed electron paramagnetic resonance (EPR) spectroscopic and kinetic studies, that in this enzyme the chromophoric FeIII can be replaced by MnII, forming a catalytically active, unprecedented antiferromagnetically coupled homodivalent MnII-µ-(H)OH-µ-carboxylato-MnII center in a PAP. However, although the enzyme is still active, it no longer functions as an acid phosphatase, having optimal activity at neutral pH. Thus, PAPs may have evolved from distantly related divalent dinuclear metallohydrolases that operate under pH neutral conditions by stabilization of a trivalent-divalent metal-ion core. The present MnII-MnII system models these distant relatives, and the results herein make a significant contribution to our understanding of the role of the chromophoric metal ion as an activator of the nucleophile. In addition, the detailed analysis of strain broadened EPR spectra from exchange-coupled dinuclear MnII-MnII centers described herein provides the basis for the full interpretation of the EPR spectra from other dinuclear Mn metalloenzymes. Introduction only exception with a confirmed heterodivalent dinuclear center III II ) 1,6,10,11 Purple acid phosphatases (PAPs) belong to the family of of the type Fe M (M Fe, Zn, Mn) is PAP. Despite a low degree of overall amino acid sequence homology and dinuclear metallohydrolases that include Ser/Thr protein phos- 12 phatases, organophosphate-degrading triesterases, ureases, ar- metal-ion content between enzymes from different kingdoms ginases, aminopeptidases, and antibiotics-degrading metallo-- their catalytic sites display a remarkable similarity. The two lactamases.1-6 Because of their widespread metabolic functions, metal ions are coordinated by seven invariant ligands (Figure 13 several of these enzymes have become targets for the develop- 1), with an aspartate residue bridging the two ions. The ment of chemotherapeutic agents or are used as bioremediators. characteristic purple color of PAPs is due to the formation of a Specifically, PAP activity in animals has been shown to be charge-transfer (CT) complex between the trivalent iron and a 14 directly linked to bone resorption; overexpression of this enzyme conserved tyrosine ligand. Animal PAPs have a redox-active by bone-resorbing osteoclasts leads to osteoporosis.7,8 Conse- quently, PAP has become a major target in the development of (7) Hayman, A. R.; Jones, S. J.; Boyde, A.; Foster, D.; Colledge, W. H.; anti-osteoporotic drugs.9 Carlton, M. B.; Cox, T. M. DeVelopment 1996, 122, 3151–3162. Virtually all dinuclear metallohydrolases have homodivalent (8) Oddie, G. W.; Schenk, G.; Angel, N. Z.; Walsh, N.; Guddat, L. W.; II II II II II II 1,6 de Jersey, J.; Cassady, A. I.; Hamilton, S. E.; Hume, D. A. Bone 2000, metal centers of the Zn Zn ,FeFe ,orMnMn type. The 27, 575–584. (9) (a) Valizadeh, M.; Schenk, G.; Nash, K.; Oddie, G. W.; Guddat, L. W.; † School of Chemistry and Molecular Biosciences. Hume, D. A.; de Jersey, J.; Burke, T. R.; Hamilton, S. Arch. Biochem. ‡ Centre of Magnetic Resonance. Biophys. 2004, 424, 154–162. (b) McGeary, R. P.; Vella, P.; Mak, (1) Wilcox, D. E. Chem. ReV. 1996, 96, 2435–2458. J. Y. W.; Guddat, L. W.; Schenk, G. Bioorg. Med. Chem. Lett. 2009, (2) Dismukes, G. C. Chem. ReV. 1996, 96, 2909–2926. 19, 163–166. (3) Barford, D.; Das, A. K.; Egloff, M. P. Annu. ReV. Biophys. Biomol. (10) Wang, D. L.; Holz, R. C.; David, S. S.; Que Jr, L.; Stankovich, M. T. Struct. 1998, 27, 133–164. Biochemistry 1991, 30, 8187–8194. (4) Lowther, W. T.; Matthews, B. W. Biochim. Biophys. Acta 2000, 1477, (11) Bernhardt, P. V.; Schenk, G.; Wilson, G. V. Biochemistry 2004, 43, 157–167. 10387–10392. (5) Crowder, M. W.; Spencer, J.; Vila, A. J. Acc. Chem. Res. 2006, 39, (12) (a) Schenk, G.; Guddat, L. W.; Ge, Y.; Carrington, L. E.; Hume, D. A.; 721–728. Hamilton, S.; de Jersey, J. Gene 2000, 250, 117–125. (b) Flanagan, (6) Mitic´, N.; Smith, S. J.; Neves, A.; Guddat, L. W.; Gahan, L. R.; J. U.; Cassady, A. I.; Schenk, G.; Guddat, L. W.; Hume, D. A. Gene Schenk, G. Chem. ReV. 2006, 106, 3338–3363. 2006, 377, 12–20. 10.1021/ja900797u CCC: $40.75 2009 American Chemical Society J. AM. CHEM. SOC. 2009, 131, 8173–8179 9 8173 ARTICLES Mitic´ et al. MnII.24-26 The precise mechanistic role(s) of the two metal ions is still subject to debate, but it has been proposed that the MII is essential in substrate binding and orientation and the FeIII activates the hydrolysis-initiating nucleophile.27-31 Herein, we have probed the role of the FeIII by substituting it with manganese. We chose a PAP from sweet potato (spPAP) as the target enzyme because it has previously been shown to require at least one Mn in the active site, forming an unprec- edented, strongly antiferromagnetically coupled FeIII-(µ-O)-MnII center under optimal catalytic conditions (Figure 1).13e,32 Experimental Section Protein Purification and Metal-Ion Replacement. spPAP was purified as described previously.25 To generate the Mn derivative, the native FeMn enzyme (0.3 mM in 0.1 M acetate buffer, pH 4.9) was incubated at room temperature (RT) with 5 mM 1,10- phenanthroline and 100 mM sodium dithionite for 20 min. The mixture was loaded ontoa3mLBio-Rad Econo-Pac 10DG column (equilibrated with 0.1 M acetate buffer, pH 4.9). Subsequently, 50 equiv of MnCl was added and the mixture was allowed to stand 13e 2 Figure 1. Schematic illustration of the active site of FeMn-spPAP. at RT for several days until maximal kinetic activity was reached His201, His295, and Glu365 assist in substrate binding and orientation. (∼100 h). The excess of manganese was then removed by dialysis The substrate analogue (PO4) is shown in a possible transition state whereby the bridging oxygen acts as a nucleophile. against 0.1 M acetate buffer, pH 4.9. The metal-ion composition was determined in triplicate using atomic absorption spectroscopy FeIIIFeIII/II center, where only the mixed-valent form is believed (Varian SpectrAA 220FS instrument). 15 III II Kinetics. The rates of product formation were determined at 25 to be catalytically competent. Reduction of the active Fe Fe ° II II C using a standard continuous assay with para-nitrophenylphos- center to Fe Fe leads to inactivation and loss of one or both p- ) 16,17 phate ( NPP) as substrate (λ 390 nm; the ∆ε was determined metal ions depending upon exposure times to the reductant. at each pH by allowing 5 mM p-NPP to hydrolyze to completion). In fact, this approach has been successfully employed to generate Measurements were carried out at various pH values ranging from - metalloderivatives of PAPs.16 23 For instance, the FeII in pig pH 3.5 to pH 9.0 (using 0.1 M glycine, acetate, 2-(N-morpholino)- PAP (uteroferrin) can be replaced by ZnII without a significant ethanesulfonic acid, and tris(hydroxymethyl)aminomethane buffers, change in activity, whereas the MnII,NiII,CuII, and CoII respectively, each with 0.2 M KCl) using a Cary 50 Bio Spectro- derivatives display 10-50% activity of the native enzyme.19,21-23 photometer. Substrate concentrations ranged from 0.25 to 10 mM, and [PAP] was 2 nM. The pH dependence of the kcat and kcat/KM Although the divalent ion can be replaced, there are no reports 22,33 of catalytically active dinuclear homodivalent PAPs. In most values were fitted to eqs 1 and 2 as described elsewhere. plant PAPs the divalent metal ion (MII) is either ZnII or ) + - + - kcat kcat1 ({(kcat4 kcat1)Kes1Kes2Kes3 (kcat3 (13) (a) Stra¨ter, N.; Klabunde, T.; Tucker, P.; Witzel, H.; Krebs, B. Science + + - + 2 + 3 + kcat1)Kes1Kes2[H ] (kcat2 kcat1)Kes1[H ] }/{[H ] 1995, 268, 1489–1492. (b) Klabunde, T.; Stra¨ter, N.; Frohlich, R.; + 2 + + + Witzel, H.; Krebs, B. J. Mol. Biol. 1996, 259, 737–748. (c) Lindqvist, Kes1[H ] Kes1Kes2[H ] Kes1Kes2Kes3}) (1) Y.; Johansson, E.; Kaija, H.; Vihko, P.; Schneider, G. J. Mol. Biol. 1999, 291, 135–147. (d) Uppenberg, J.; Lindqvist, F.; Svensson, C.; Ek-Rylander, B.; Andersson, G. J. Mol. Biol. 1999, 290, 201–211. where Kesn (n ) 1, 2, 3) represents a protonation equilibrium of (e) Schenk, G.; Gahan, L. R.; Carrington, L. E.; Mitic´, N.; Valizadeh, the enzyme-substrate complex and kcatn (n ) 1, 2, 3, 4) corresponds M.; Hamilton, S. E.; de Jersey, J.; Guddat, L. W. Proc. Natl. Acad. to the activity of the associated protonation states. Sci. U.S.A. 2005, 102, 273–278.
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