Substrate positioning by His92 is important in catalysis by purple acid phosphatase Enrico G. Funhoff 1,4 , Yunling Wang 2 , Goran Andersson 2 and Bruce A. Averill 1,3 1 Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands 2 Karolinska Institutet, Division of Pathology, Huddinge University Hospital, Sweden 3 Department of Chemistry, University of Toledo, OH, USA 4 Institute of Biotechnology, HPT, ETH Ho ¨ nggerberg, Zu ¨ rich, Switzerland The binuclear metalloenzyme purple acid phosphatase (PAP) [1], which may be involved in disorders such as osteoporosis [2–4], Gaucher disease [5], hairy cell leukemia [6], and AIDS [7] is widely distributed in mammalian tissues [8,9]. The expression level of PAP [also referred to as tartrate resistant acid phosphatase (TRAP) or type 5 acid phosphatase (AcP5; EC 3.1.3.2)] is elevated in these disorders, suggesting a relationship between the increased levels of the enzyme and the clinical picture. The presumed role of PAP makes it important to develop drugs that can inhibit PAP activity. In order to facilitate this process, the precise catalytic mechanism must be elucidated, inclu- ding the function of each residue involved in catalysis. Although the available crystal structures of PAPs [10–12] provide structural information about the resi- dues potentially involved in catalysis, to date only structures of inactive redox and protonation states of the enzymes have been reported. All structures show an active site composed of two metal ions bridged by a solvent-derived species and a bidentate aspartate resi- due. In addition, the iron(III) ion is coordinated by a tyrosinate [resulting in a ligand-to-metal charge trans- fer (LMCT) transition that is responsible for the Keywords kinetics; mechanism; mutagenesis; purple acid phosphatase; spectroscopy Correspondence B. A. Averill, Department of Chemistry, University of Toledo, 2801 West Bancroft Road, Toledo, Ohio 43606–3390, USA Fax: +1 419 5301586 Tel: +1 419 5301585 E-mail: baa@utoledo.edu Website: http://www.chem.utoledo.edu/ FAC_INFO/Bruce/SOURCE.htm (Received 12 January 2005, revised 13 March 2005, accepted 28 March 2005) doi:10.1111/j.1742-4658.2005.04686.x Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and ⁄ or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypep- tide PAP. Conversion of His92 into Ala, which eliminates polar interac- tions of this residue with the active site, resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen–bonding interactions, resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more aci- dic pH optima, with pK es,1 values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal. Abbreviations kPP, protein phosphatase from phage k; KBPAP, kidney bean PAP; LMCT, ligand-to-metal charge transfer; MOI, multiplicity of infection; PAP, purple acid phosphatase; p-NPP, para-nitrophenylphosphate; PP, protein phosphatase; recHPAP, recombinant human purple acid phosphatase; recRPAP, recombinant rat PAP; TRAP, tartrate resistant acid phosphatase. 2968 FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS purple color], a histidine, and an aspartate residue. The site that contains the iron(II) ion in the active enzyme is coordinated by two histidine residues and an asparagine. Very recently, a structure of recombinant human PAP expressed in Escherichia coli was reported that is very different from the previous structures, in that Asp147 of the repression loop, rather than a phos- phate ion, coordinates to the dinuclear metal center in a bidentate bridging mode [13]. The above-mentioned ligating residues are conserved in several other phosphatases [14], including the closely related protein phosphatases (PPs). As shown by sev- eral crystal structures [15–19], however, PPs lack the tyrosinate coordinated to the iron(III) ion; in PPs, the tyrosinate is effectively replaced by a solvent-derived ligand to the iron(III) ion. Because of the similarity of their active sites, the catalytic mechanisms of PAPs and PPs are believed to be similar [20]. The lack of systematic studies involving the metal-coordinating res- idues in PAPs makes it difficult to interpret the func- tions of these residues. In contrast, several site-directed mutagenesis studies have been performed with PPs, which makes them potentially valuable for understand- ing the PAP mechanism. Three residues in both the PAPs and the PPs have been proposed to be involved in substrate binding: the metal coordinating Asn91, and the nonmetal coordina- ting His195 and His92 (numbering is according to the human PAP sequence [21]). Mutagenesis of His195 to alanine in kidney bean PAP (KBPAP) or to alanine and glutamine in recombinant rat PAP (recRPAP) resulted in a sharp decrease in activity [22,23]. Consequently, Asn91 was suggested to be involved in the activation of PAP, as well as in coordinating phosphate [24]. In PPs and KBPAP, His92 is part of a histidine ⁄ aspartate pair, which together with a solvent molecule could be analogous to the catalytic aspartate ⁄ histi- dine ⁄ serine triad of serine proteases [25]. In principle, His92 could function as: (a) an active site nucleophile; (b) a general acid catalyst that protonates the leaving group; or (c) a general base that deprotonates a sol- vent molecule coordinated to iron [25]. Because direct transfer of phosphate to water is observed and k cat is independent of the pK a of the leaving group, options (a) and (b) are highly unlikely. Isotope effect studies on k phage PP (kPP) [26], however, showed no clear evidence for option (c). Moreover, the Asn mutant of His76 in kPP showed a basic limb in the pH optimum that should not have been present [26]. A fourth poss- ible role for His92, which is as yet unexplored, is posi- tioning of the nucleophilic hydroxide or substrate for optimal in-line attack [20]. Thus, the precise role of His92 is still unclear. To further study the role of His92 and its interaction with Asp146 in PAP, we have prepared mutants of this residue and characterized their kinetics and spectro- scopic properties. The characteristics of the His92Asn and His92Ala mutants do not support the proposal that His92 is involved in the catalytic process as either a proton donor for the leaving group or as a base that regenerates the nucleophilic hydroxide. They do, how- ever, suggest an important role for this residue in posi- tioning of either the substrate or the nucleophile. Results Production of single polypeptide mutant recRPAP (His92Asn, and His92Ala) in 1 L shaking flask cultures resulted in good yields of the His92Asn and His92Ala mutant enzymes. Purification of the mutant enzymes gave protein samples with A 280 ⁄ A kmax values of 17–20 (for pure PAP A 280 ⁄ A kmax % 16), and a single band with small impurities (% 5%) was observed for each mutant in SDS ⁄ PAGE gels stained with Coomassie brilliant blue. Kinetics characteristics of mutant enzymes In order to examine the pH dependence of the single polypeptide mutants, the pH optimum was measured at a single substrate concentration [50 mm para-nitro- phenylphosphate (p-NPP)] for both mutants. The pH optima obtained with this procedure differ slightly from the actual pH optima (as determined from a plot of k cat vs. pH), due to nonsaturating substrate condi- tions at higher pH values [27]. Because unexpected val- ues for k cat were observed and because K M was high at the optimal pH, subsequent measurements of k cat vs. pH were performed (Fig. 1). The k cat vs. pH plot of the His92Ala mutant was analyzed according to the rapid equilibrium diprotic model [28] to give the values of pK es presented in Table 1. The k cat vs. pH plot showed a broad optimum at pH 3.8, which is significantly shifted compared to native recombinant RPAP [24], with apparent pK es val- ues of 2.6 and 5.2 (Fig. 1). The pH optimum and pK a values obtained by fitting the data have an error of approximately ± 0.2 pH units. Because of the low activity, incubation times during the assay were increased (from 1 to 5 min), but even at the lowest pH the enzyme was stable over the time range measured. Although the k cat values have rather small errors, the plot of K M vs. pH suffers from rather large error bars at each pH. Apparently K M parallels the behavior of k cat as a function of pH, rather than simply increasing with increasing pH. Because of this behavior, k cat ⁄ K M E. G. Funhoff et al. Mutational analysis of His92 in recombinant rat PAP FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS 2969 does not show the typical sigmoidal curve from which apK e2 value can be determined, as is found for the native enzyme. The maximal fitted value for k cat at pH 3.8 is 21 s )1 , less than 10% that of wild-type enzyme at its optimal pH, while K M is 15- to 30-fold larger compared to the wild-type at pH 4. The value of k cat reported in Table 1 is that obtained by fitting the data; the apparent maximal k cat in Fig. 1 is lower due to the small difference between the two pK a values. Fig. 1. Plots of k cat , k cat ⁄ K M and K M vs. pH for single polypeptide recRPAP (upper panel, adapted from [24]), His92Ala-recRPAP (middle panel) and His92Asn-recRPAP (lower panel) with p-NPP as substrate at 22 °C. The lines represent fits of the data to the rapid equilibrium diprotic model. The following expression was derived for the observed values of k cat : k cat(obs) ¼ k cat ⁄ (1 + [H + ] ⁄ K es,1 + K es,2 ⁄ [H + ]); assuming that all equilibria are fast compared to k cat , k cat(obs) ⁄ K M(obs) ¼ k cat ⁄ K S (1 + [H + ] ⁄ K e1 + K e2 ⁄ [H + ]). K S is the dissociation constant of the enzyme–substrate complex. Mutational analysis of His92 in recombinant rat PAP E. G. Funhoff et al. 2970 FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS The His92Asn mutant shows an optimum at pH 4.4, with pK es values of 3.2 and 5.6 (Fig. 1) and a turnover number of approximately 760 s )1 , three times higher than that of the wild-type recRPAP. K M increases with increasing pH, and is about 10 times larger than that of the wild type at pH 4.4. Analysis of the k cat ⁄ K M plot shows that values for pK e,1 and pK e,2 can be fitted but the large error bars suggest that these data should be interpreted with some caution. A value of approxi- mately 2.2 can be derived from the fits for pK e,1 , while for pK e,2 a value of 4.9 is found. Spectroscopic characteristics of mutant enzymes The EPR spectrum of the fully reduced native recR- PAP shows different species at different pH values (Fig. 2A). At pH 7, which is above pK es,2 , a signal with features at g xyz ¼ 1.58, 1.74, 1.97 is observed. Upon decreasing the pH to the optimal pH 5.5, a spe- cies with g xyz ¼ 1.59, 1.74, 1.93 is formed, while a third species is formed at pH values below pK es,1 , with g xyz ¼ 1.60, 1.74, 1.86. This behavior is very similar to the pH dependency of the EPR spectrum of recombin- ant human purple acid phosphatase (recHPAP), which is reported in detail in [29]. The His92Ala mutant shows the presence of a single species at its pH optimum (Fig. 2B), with apparent g-values of g xyz ¼ 1.58, 1.73, 1.97, corresponding to the species that is observed above pK es,2 of the native enzyme. Measuring the spectrum at pH ¼ pK es,1 was not possible, due to the instability of the mutant enzyme during buffer exchange. Increasing the pH to pH ¼ pK es,2 gave no change in the EPR spectrum. Thus, for the His92Ala mutant, a single EPR detect- able species is present over the pH range 3.7–6.5. The intensity of the signal due to this species is reduced at pH 4.1 and 3.7 to % 0.5–0.7 and % 0.1–0.15 spins per Table 1. Kinetics parameters of single polypeptide His92Asn, and His92Ala mutants of recRPAP. k cat is defined as the number of substrate molecules hydrolyzed per enzyme molecule per second; pK es,1 and pK es,2 are for deprotonation ⁄ protonation events of a group of the enzyme–substrate complex; and pK e is for a deproto- nation ⁄ protonation event of a group of the free enzyme. All pK val- ues reported have an error of ± 0.2 pH units. pH opt pK es,1 pK es,2 pK e,1 pK e,2 k cat (s )1 ) K M a (mM) RecRPAP b 5.5 4.5 6.6 – 5.6 240 4.5 His92Asn 4.4 3.2 5.6 2.2 4.9 760 23 His92Ala 3.8 2.6 5.2 – – 21 35 a At the optimal pH. b From [24]. Fig. 2. EPR spectra of native recRPAP (A), His92Ala-recRPAP (B) and His92Asn-recRPAP (C) at different pH values. The spectrum in the lower panel of Fig. 2C is a five-fold enlargement of the pH 2.7 spectrum. The inset in Fig. 2C shows the superposition of two simulated spectra (solid black line) with g values of g xyz ¼ 1.57, 1.70, 1.85 (ÆÆÆÆ), and g xyz ¼ 1.41, 1.60, 1.74 (dashed line) together with the measured spectrum (solid gray line). EPR conditions: (A) microwave power 2 mW; microwave frequency, 9.423 GHz; modulation, 12.7 G at 100 kHz; temperature, 4.5–5.5 K. All spectra in (A) were normalized for gain, temperature, and protein concentration; (B) same conditions as for (A) with a tempera- ture range 4.5–5.7 K and different power. All spectra were normalized for gain, temperature, power, and protein concentration. E. G. Funhoff et al. Mutational analysis of His92 in recombinant rat PAP FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS 2971 molecule, respectively, suggesting a correlation with the protonation of a catalytically important residue. In the visible spectra only small shifts in k max with pH are observed: for example, at pH 3.7 k max is 504 nm vs. 508 nm at pH 6.5 (data not shown). The EPR spectrum of the His92Asn mutant at pH 6.5 shows apparent g values at 1.96, 1.85, 1.71, 1.60, and 1.41 (Fig. 2C), indicating the presence of several species. Simulation of the spectra suggests that two main species are present, with features at g xyz ¼ 1.57, 1.70, 1.85, and g xyz ¼ 1.41, 1.60, 1.74, respect- ively. A weak signal with g xyz ¼ 1.58, 1.74, 1.93 could also be present. Decreasing the pH to 2.7 caused the intensity of the g xyz ¼ 1.57, 1.70, 1.85 species to decrease relative to that of the g xyz ¼ 1.41, 1.60, 1.74 species. At pH ¼ pK es,1 (2.7), the total intensity of the spectrum decreased from % 0.5 spins per molecule to < 0.05 spins. Together with the loss of signal intensity at g av ¼ 1.74, an increase in the intensity of the signal due to high-spin Fe 3+ species was observed, from % 1–2% to % 45% of the total signal intensity, respect- ively. This was not due to air oxidation, because the original spectrum was restored simply by raising the pH of the sample to 6.5. A shift of k max to higher wavelength was observed, which parallels the loss of signal intensity in the EPR spectrum: k max of the His92Asn mutant shifted from 530 nm at pH 6.5 and pH 4.4 to 560 nm at pH 2.7. Increasing the pH of the sample to pH 6.5 restored the original k max of 530 nm (data not shown). Discussion To date, two site-directed mutagenesis studies of first or second coordination sphere active site residues of PAP have been published. The function of the loop residue Asp146 in recHPAP has been extensively stud- ied [24], while preliminary kinetics results on His92 and His195 mutants have been published [23]. In the latter study, turnover numbers of 2.4 and 7.8 s )1 were meas- ured at pH 4.5 for His92Ala and His92Gln, which is 10–100-fold lower than we observe for the His92Ala and His92Asn mutants at this pH. Moreover, Michaelis-Menten constants of approximately 7–15 mm were observed at pH 4.5, although a K M of 40 mm for the wild-type enzyme was found [23]. The K M value of other single polypeptide PAPs at this pH is significantly lower, around 1 mm [24,30,31]. Several studies have appeared on the closely related PPs [14], in which all active site residues have been mutated [25,26,32–37]. In most of these studies, the kinetics parameters were determined under only one set of conditions, which did not result in a clear under- standing of the function of these residues. Studies by the Rusnak group have provided more detailed insight into the role of several active site residues [25,26,37]. Two of the corresponding residues in PAPs, Asn91 and His92, have been proposed to be involved in pro- teolytic activation of mammalian FeFe-PAPs due to a possible interaction with the loop residue Asp146 [24,30]. The interaction of these two residues with Asp146 in an exposed loop reduces the catalytic activ- ity and decreases pK es,1 [24] which is the pK a of the metal-coordinated solvent molecule [29]. In the present study, the His residue that could interact with Asp146 has been mutated to elucidate its role in the catalytic process. Mertz et al. [25] suggested that His92 in PPs is not required for protonation of the leaving group, because the same relative k cat value was found for two sub- strates with different leaving group pK a values. Instead, they suggested that His92 could function in concert with the nucleophilic water molecule to either position a lone pair on the oxygen atom for optimum in-line attack on the phosphorus atom of the substrate or serve as a general base to take up a proton concom- itant with solvent nucleophilic attack. A subsequent isotope effect study of the wild-type and His76Asn- kPP showed that the increase in [ 15 N](V ⁄ K) and [ 18 O](V ⁄ K) bridge isotope effects for the substrate p-NPP were analogous to those observed for protein tyrosine phosphatases observed upon mutation of their general acid, but smaller in magnitude. Thus, these studies did not clearly answer the question of whether His76 func- tions as a general base [26]. For recRPAP, the pH optima of both the His92Asn and His92Ala mutants are shifted 1–1.5 pH units to lower values due to a 1.5–2 pH unit shift in pK es,1 ;in addition pK es,2 decreases by one pH unit. By compar- ison, the pH optimum of His76Asn-kPP shows more than a full pH unit decrease [26]. Although it has been suggested more than once that pK es,2 is due to depro- tonation of the His92 imidazole group, the presence of a basic limb in the pH profiles of mutants of three enzymes with related active site structures (His92Asn- recRPAP, His76Asn-kPP [26], and His92Ala-recRPAP) provides convincing evidence that pK es,2 is not due to the (de)protonation of the imidazole group of His92. The large (15- to 30-fold) increase in K M observed for His92Ala-recRPAP suggests that this residue is involved in substrate binding in PAPs. In principle, however, an Asn residue at position 92 should be able to hydrogen bond to the substrate, but this mutant also shows significantly increased values for K M , argu- ing against such a role in substrate binding. Previous site-directed mutagenesis studies of kPP also argue Mutational analysis of His92 in recombinant rat PAP E. G. Funhoff et al. 2972 FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS against such a role, although these results can also be explained in favor of monoanion binding [25,26]. For all the PP mutants, k cat was ¼ 1% of that of the wild- type enzyme, in dramatic contrast with the observed k cat values for His92Ala and His92Asn-recRPAP [25,26,32,34]. The almost three-fold increase in k cat of the His92Asn mutant compared to the wild-type enzyme is particularly surprising. The shift of pK a,1 to lower pH upon mutation of His92 into Ala and the increase in k cat for the His92Asn mutant strongly sug- gest that the presence of the His92 imidazole group increases pK a,1 (the pK a of the nucleophile), but does not significantly affect its reactivity. Thus, His92 does not function as an acid or a base in the catalytic cycle, either in regeneration of the nucleophile as has been proposed for metal-containing enzymes such as argi- nase or carbonic anhydrase [38], or by abstracting or donating a proton to the substrate or product. It is clear, however, that His92 does play a major role in enzyme catalysis. Although detailed mechanistic studies are lacking, the congruence of active site structures strongly sug- gests that PAPs and PPs catalyze hydrolysis of phos- phate ester substrates via very similar mechanisms [20]; in particular, it seems very likely that the groups responsible for pK a,1 and pK a,2 are identical in the two sets of enzymes. The group responsible for pK a,1 is a metal-bound solvent molecule whose identity is unknown [29,39]. Possibilities include a terminally coordinated Fe 3+ or Fe 2+ hydroxide, a bridging hydroxide ion, or a second coordination sphere hydroxide [40,41]. Observed values of pK a,1 for PAP range from 5.5 for the proteolytically cleaved wild-type enzyme to 4.5 for wild-type single polypeptide [24,30] to 2.6 for the His92Ala mutant. Based on the pK a values of hexa-aquo complexes of metal ions [42,43], it is not feasible to attribute a pK a,1 of 2.6 (His92Ala) or 3.2 (His92Asn) to a water group coordinated to a divalent transition metal ion. Although the most plausible assumption is that pK a,1 is due to a solvent molecule coordinated terminally to the trivalent ion, there are very strong arguments against a terminal trivalent coordinated nucleophile; they include the following: (a) Replacement of the trivalent metal ion by other metals has essentially no effect on the kinetics proper- ties, while substitution of the divalent site results in significant kinetics changes [44,45]. The involvement of the divalent site is most pronounced for single poly- peptide recHPAP, where an increase in k cat from 210 to 5000 s )1 is observed for FeZn-recHPAP. Further- more, proteolysis does not result in activation or a sig- nificant change in pK es,1 and pK es,2 [46]. (b) Fluoride can replace the hydroxide only if it is protonated [29]. Moreover, the disappearance of the NMR spectrum of the enzyme–fluoride complex below pK es,1 is consistent with a significant change in the super-exchange interaction between the two iron ions, suggesting that fluoride replaces a solvent molecule bridging both metal ions [27]. (c) ENDOR results on single polypeptide uteroferrin could not detect a water-derived ligand coordinated to the trivalent site [41]. Thus, many results point in the direction of a nucle- ophilic hydroxide that is coordinated to the divalent metal ion, which is difficult to correlate with the pK es,1 values observed for the His92Asn and His92Ala mutants. One possibility that could resolve this contro- versy is the possibility that a solvent molecule in the second coordination sphere acts as the nucleophile [45]. The EPR spectrum of native recRPAP shows the presence of three different species at different pH values, and these are also observed for recHPAP. For recHPAP the change in the EPR spectrum from a feature with g xyz ¼ 1.58, 1.74, 1.94 to a feature with g xyz ¼ 1.58, 1.74, 1.97 correlates well with pK es,2 [29]. The only species observed by EPR for His92Ala-recRPAP over the pH range 4–8 resembles native recRPAP and recHPAP at pH > pK es,2 . The intensity of this species is reduced at pH 3.7 and 4.1 compared to the spectrum at pH 6.5. This suggests that His92 is apparently capable of interacting with the metal site in PAP (and, presumably, in kPP and PP1) either directly or via solvent molecules, as sug- gested by the structures of uncomplexed forms of PP1 [16] and calcineurin [15], even though it is 4.5– 5A ˚ away from the metals in PAPs [11,12,47] and PPs [17–19]. Thus, it could well be that in the wild- type enzyme at pH ¼ pK a,2 a change in the position of His92 is responsible for the observed shift in the g z feature of the EPR spectrum (from 1.93 to 1.97). Because mutagenesis has shown that His92 itself is not responsible for pK es,2 , deprotonation of an as yet unidentified residue that interacts with His92 might force the latter into a different position, one that is less favorable for catalysis. To gain better insight into the structural effects of the mutations, first approach models with energy minimization were obtained using SwissModel and DeepView ⁄ Swiss PdB-viewer. First approach mode- ling of the His92Asn mutant (Fig. 3) shows that the amido nitrogen of Asn92 does not coordinate to the phosphate ion, but points in the direction of Asp146. A weak interaction could be present, jud- ging from the distance of 3.34 A ˚ between the Asn92 E. G. Funhoff et al. Mutational analysis of His92 in recombinant rat PAP FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS 2973 amido nitrogen and the Asp146 carboxylate, which could weaken the Asp146–Asn91 interaction that decreases the Lewis acidity of the iron(II) ion and thereby the electrophilicity of the activated substrate molecule [24,30]. This hypothesis would explain the higher activity of the His92Asn mutant, but it does not account for the lower pK es values. In conclusion, the observed low pK es values sup- port a model in which the nucleophilic hydroxide is that coordinated to the iron(III) ion and whose reac- tivity is tuned by the position of His92. However, the dramatic effects of metal substitution on the iron(II) site [46], together with the fluoride inhibition ⁄ spectroscopic studies [27] suggest a bridging hydr- oxide as nucleophile. Hypothetically, the flexible character of the bridging hydroxide [48] and its inter- action with His92 could result in [partial] opening of the hydroxide bridge, resulting in an equilibrium between two species: one in which the hydroxide interacts more strongly with the divalent metal ion, resulting in higher pK es values and a higher k cat due to faster ligand exchange rates: and one in which it interacts more strongly with the trivalent metal, resulting in more acidic pK es values and lower k cat values. High-resolution crystal structures of a mam- malian PAP in active redox and pH states in the presence and absence of nonhydrolysable substrate analogues, such as AMP, perhaps in combination with ENDOR spectroscopy, will be necessary to elu- cidate the mechanism of PAPs. Experimental procedures General procedures Enzyme concentrations were determined by measuring the absorbance of the Tyr – to-Fe 3+ charge transfer at k max (510–550 nm; e ¼ 4080 m )1 cm )1 ) [49] on a Cary 50 or HP8452A photodiode array spectrophotometer (Varian Inc., Palo Alto, CA, USA). Generation of mutant proteins RecRPAP mutant enzymes were prepared as previously described with the QuickChange TM Site-Directed Mutagen- esis Kit (Stratagene, La Jolla, CA, USA) [24]. Primers used for specific mutations were as follows: His92Ala, 5¢-CTGG CTGGAAAC GCTGATCACCT TGGC-3¢; His92Asn, 5¢-GGC TGGAAAC AATGATCACCTTG-3¢. The underlined bases indicate changes compared to the wild-type sequence. Recombinant baculovirus stocks containing regions coding for the His92Asn, and His92Ala mutants were used to infect High 5 cells cultured in 500–1000 mL Excel 405 TM SFM at 27 °C; the cell density was 0.7–0.9 · 10 6 cellsÆmL )1 , and a low multiplicity of infection (MOI; 0.001–0.01) was used. After 5 days the cells were removed Fig. 3. Active site structure of phosphate-complexed uteroferrin at 1.55 A ˚ resolution (left panel) showing the hydrogen bonding network between the divalent metal (II), trivalent metal (III), Asn91, and His92. The distances between the residues are given in A ˚ . The backbone of His92 is stabilized via hydrogen bonds between the amido nitrogen of Asn144 and the backbone carbonyl of Asn91, and between the carb- oxylate of Asp52 and the backbone nitrogen of His92. The interaction between the amido nitrogen of Asn91 and the carboxylate of Asp146 and the intraloop hydrogen bond between the Ser145 backbone amino group and the side chain oxygen of Asn144 further fine-tune this hydrogen bonding network. The right panel shows the first approach modeling structure of the His92Asn mutant after energy minimization. The amido nitrogen of Asn92 is oriented towards Asp146 and shows weak interaction with the Asp146 carboxylate group. The figure was generated using the D EEPVIEW ⁄ SWISS-PDBVIEWER program with the coordinates of 1UTE. Mutational analysis of His92 in recombinant rat PAP E. G. Funhoff et al. 2974 FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS by centrifugation (10 000 g), and the fully reduced enzyme was purified from the medium as previously des- cribed [24]. Kinetics measurements The pH dependence of the catalytic activity of His92Asn and His92Ala was measured in 100 mm buffer (sodium acetate, Mes and Hepes), 300 mm KCl, 10 mm Na ⁄ K tar- trate, 6.7 mm sodium ascorbate, 0.37 mm Fe(NH 4 ) 2 (SO 4 ) 2 , and substrate concentrations between 1 and 100 mm p-NPP as previously described [24]. At intervals after enzyme addi- tion, 250 lL aliquots were removed and quenched with 1.0 mL of 0.5 m NaOH to convert all product to the phe- nolate form. For each determination of V max and K M , the hydrolysis rate was measured using at least six different p-NPP concentrations with assay times between 1 and 5 min. Enzyme concentrations were varied to ensure a sig- nificant change in absorbance. After each assay, the pH of the reaction mixture was measured to ensure that it had not changed. Values of K M and V max were obtained by fit- ting the data to the Michaelis–Menten equation: v ¼ðV à max ½SÞ=ðK M þ½S Þ using the program Leonora (Athel-Cornish Bowden, ver- sion 1). The pH dependencies of values of k cat vs. pH were ana- lyzed according to the rapid equilibrium diprotic model [28], which is used if the difference in pK a values is less than 3.5 pH units. The following expressions were derived for k cat and k cat ⁄ K M : k catðobservedÞ ¼ k cat =ð1 þ½H þ =K es;1 þ K es;2 =½H þ Þ k catðobsÞ =K MðobsÞ ¼ k cat =K S ð1 þ½H þ =K e1 þ K e2 =½H þ Þ: Electron paramagnetic resonance (EPR) spectroscopy Samples of fully reduced native and mutant recRPAP were prepared in a buffer of the appropriate pH [100 mm sodium acetate, Mes, Hepes; 0.45 m KCl, 20% (v ⁄ v) glycerol], and frozen in liquid N 2 . X-band EPR spectra (9.43 GHz) were obtained on a Bruker ECS106 EPR spectrometer (Bruker-Biospin Corp., Billerica, MA, USA) equipped with an Oxford Instruments ESR900 helium-flow cryostat with an ITC4 temperature controller. The magnetic field was calibrated with an AEG Magnetic Field Meter, and the frequency was measured with a HP 5350B Micro- wave Frequency Counter. After recording the spectrum, the sample was thawed and buffer exchanged into a buffer with the appropriate pH by repetitive concentration ⁄ dilution using a Microcon (30 kDa cut-off). Complete buffer exchange during the complete EPR experiment took several hours. Acknowledgements We thank Robert-Jan Sanders for his initial work on the mutant enzymes. This research was supported by grants from the Swedish Research Council (to GA) and the Research Funds of Karolinska Institutet, and by a contract from the EU 4th Framework Biotechno- logy Program (BI04-CT98-0385). References 1 Averill BA (2003) Dimetal hydrolases. 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J Am Chem Soc 119, 11832–11842. 49 Davis JC, Lin SS & Averill BA (1981) Kinetics and optical spectroscopic studies on the purple acid phos- phatase from beef spleen. Biochemistry 20, 4062–4067. E. G. Funhoff et al. Mutational analysis of His92 in recombinant rat PAP FEBS Journal 272 (2005) 2968–2977 ª 2005 FEBS 2977 . Substrate positioning by His92 is important in catalysis by purple acid phosphatase Enrico G. Funhoff 1,4 , Yunling Wang 2 , Goran Andersson 2 and. of His92. The large (15- to 30-fold) increase in K M observed for His92Ala-recRPAP suggests that this residue is involved in substrate binding in PAPs. In