Tài liệu Báo cáo khoa học: Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus pdf

Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus Yifat F. Hershcovitz 1 , Rotem Gilboa 2 , Vera Reiland 2 , Gil Shoham 2 and Yuval Shoham 1 1 Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa, Israel 2 Department of Inorganic Chemistry, The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Israel Aminopeptidases are exopeptidases that catalyze the removal of N-terminal amino acids from peptides; they are found in bacteria, plants and mammalian tissues. Many aminopeptidases are metallo-enzymes, containing two catalytic transition metals (usually zinc) in their act- ive site [1–3]. The activity of these enzymes is associated with many central biological processes, such as protein maturation, protein degradation, hormone level regula- tion, angiogenesis and cell-cycle control [4–8]. Not surprisingly, aminopeptidases play an important role in many pathological conditions, including cancer, cata- ract, cystic fibrosis and HIV infection. Indeed, anti- tumor drugs such as ovalicin and fumagillin were found to inhibit aminopeptidases. In this regard, the natural inhibitor for aminopeptidases, bestatin, was recently shown to significantly decrease HIV infection by inhibit- ing aminopeptidase activity [9–11]. Aminopeptidases can be classified into clans and families based on their amino acid sequence homology. Clan M contains mainly metallopeptidase families, one of which is M28. Keywords aminopeptidase; catalytic mechanism; catalytic residues; fluoride inhibition; isotope effect Correspondence Y. Shoham, Department of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel Fax: +972 4 8293399 Tel: +972 4 8293072 E-mail: yshoham@tx.technion.ac.il (Received 30 April 2007, revised 28 May 2007, accepted 1 June 2007) doi:10.1111/j.1742-4658.2007.05912.x The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH-dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine-para-nitroanilide by SGAP. The value obtained (30 ± 5 kJÆmol )1 ) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water ⁄ hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure non- competitive inhibitor of SGAP at pH values of 5.9–8 with a K i of 11.4 mm at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme–substrate complex. pH-dependent pK i experi- ments resulted in a pK a value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid–base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (K m , k cat ) over a tempera- ture range of 293–329 °K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resul- ted in a four orders of magnitude decrease in k cat , corresponding to an increase of 9 kJÆmol )1 in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis. Abbreviations AAP, Aeromonas proteolytica aminopeptidase; blLAP, bovine lens leucine aminopeptidase; Leu-pNA, leucine-para-nitroanilide; SGAP, Streptomyces griseus aminopeptidase. 3864 FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS Family M28 is currently divided into five subfamilies, M28A–M28E [12,13]. The M28 family includes several bacterial aminopeptidases, such as M28A Streptomyces griseus aminopeptidase [(SGAP) EC 3.4.11.10] and M28E Aeromonas proteolytica aminopeptidase [(AAP) EC 3.4.11.10]. In addition, the M28 family includes important human aminopeptidases such as M28B glu- tamate carboxypeptidase II (N-acetylated, alpha-linked acidic dipeptidase, prostate-specific membrane antigen) [9,13–23]. The crystal structure of several double-zinc aminopeptidases has been determined, including that of SGAP, AAP [24–30] and the bovine lens leucine ami- nopeptidase [(blLAP) EC 3.4.11.1] [31–34]. Based on biochemical and structural data, a general catalytic mechanism was proposed for aminopeptidases that involves an acidic residue acting as a general acid ⁄ gen- eral base and a di-nuclear metal center participating in binding the substrate and stabilizing the transition state [2,14,35–37]. The main data presently available for aminopeptidases and their catalytic mode of action are summarized in several recent reviews [2,14,38]. SGAP is a monomeric (30 kDa) thermostable enzyme that prefers large hydrophobic amino-terminus residues in its peptide and protein substrates. This enzyme contains two zinc ions in its active site and was shown to be activated by calcium ions [39,40]. High-resolution crystal structures of SGAP and com- plexes of the enzyme with reaction products were determined [26–28] and used together with biochemical data from SGAP and other double-zinc aminopeptid- ases [2,14] in postulating a general catalytic mechanism for this enzyme [27]. Recently, the SGAP gene was cloned and expressed in Escherichia coli, enabling researchers to verify, by site-directed mutagenesis, the role of two main catalytic residues, Glu131 and Tyr246 [36,41]. It was suggested that the acidic residue (Glu131 in SGAP corresponding to Glu151 in AAP) acts as a general base and generates the hydroxide nucleophile from the zinc-bound water; the nucleophile then attacks the carbonyl carbon of the target peptide bond, leading to the formation of a gem-diolate inter- mediate. Presumably, the abstracted proton is trans- ferred by the acidic residue (Glu131) to the leaving peptide amine group, resulting in the breakdown of the intermediate. The second catalytic residue, Tyr246, which so far was shown to be critical only in SGAP, can form hydrogen bonds with the substrate carbonyl oxygen and thus can stabilize the interaction between this oxygen atom and one of the zinc ions in the active site (Fig. 1) [2,14,27,42]. SGAP and AAP were shown to be quite similar in size, sequence, thermostability and overall structure. Nevertheless, a number of significant features differ- entiate these apparently homologous enzymes, sug- gesting that their exact catalytic mechanisms (and probably those of the corresponding subfamilies, M28A and M28E) are not completely identical. The most significant differences between these two enzymes Fig. 1. The proposed catalytic mechanism of SGAP. An acidic residue (Glu131) activates a zinc-bound water molecule and an addi- tional residue (Tyr246) polarizes the carbonyl carbon and stabilizes the transition state. Glu131 is thought to act as a general base and to generate the hydroxide nucleophile from the zinc-bound water; the nucleophile then attacks the carbonyl carbon of the tar- get peptide bond leading to the formation of a gem-diolate intermediate. The abstracted proton is presumably transferred by the aci- dic residue (Glu131) to the amine group of the leaving peptide bringing to the break- down of the intermediate. Dashed lines indi- cate stabilizing interactions and ⁄ or hydrogen bonds in the catalytic pathway; Pep, the incoming peptide ⁄ protein substrate. Y. F. Hershcovitz et al. Mechanism of an aminopeptidase from S. griseus FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS 3865 are that: (a) AAP is almost fully active (approximately 80%) [14,35,43–45], with only one zinc ion in the active site, whereas the corresponding SGAP was shown to be approximately 50% active, with 1 mol of Zn 2+ per mol of enzyme [39]; (b) the activity of SGAP is modulated by calcium ions bound in two specific sites, whereas AAP does not bind Ca 2+ [10,28,46]; (c) in AAP, there is no homologues residue to the SGAP catalytic residue, Tyr246 [36]; (d) the binding affinities to the natural inhibitors bestatin and amastatin are approximately two-fold larger in AAP than in SGAP [10]; and (e) in SGAP, the free amine group of the substrate forms strong interactions with three protein residues near the active site, whereas in AAP the free amine interacts with the second zinc ion (Zn2) [24–28]. Open issues regarding the catalytic mechanism underlying SGAP include the exact binding mode of the hydroxide to the metal ions, the proton pathway in catalysis and the specific involvement of the catalytic residues in the enzymatic reaction. The two zinc ions in the active metal center are thought to participate in substrate binding by activating the water ⁄ hydroxide nucleophile and stabilizing the transition state. Specif- ically regarding SGAP, whether the water ⁄ hydroxide molecule becomes terminally bound (bound to a single zinc molecule) during the reaction pathway remains unclear. In their biochemical studies on SGAP, Harris and Ming [47] proposed that the bridging hydroxide undergoes a single interaction at some point of the cat- alytic reaction. A similar conclusion was derived for the catalytic mechanism of AAP, in which the bridging water molecule was thought to become terminally bound following substrate binding [35]. This was based on several lines of experimental evidence: (a) 80% AAP activity was obtained with a single Zn ion bound; (b) the mode of inhibition of external anions; and (c) EPR data observed in the presence of the inhibitor butane boronic acid [35,48]. However, according to recent crystal structures of SGAP and its complexes, it is suggested that the water ⁄ hydroxide molecule could be maintained by the two zinc ions along the reaction pathway [49], without traversing terminally bound water ⁄ hydroxide species. A similar situation was pro- posed for the hexameric aminopeptidase blLAP, based on its crystal structure in complex with a transition state analog [33,34]. In the present study, we utilized the inhibition by external anions to study the binding mode of the hydroxide ⁄ water molecule in the SGAP metal center. In addition, proton transfer during catalysis was assessed by measuring the isotope effect at different pHs, for the native enzyme and its catalytic mutant E131D. The exact mechanistic role of Glu131 was explored by analyzing the temperature dependence of the kinetic parameters. Interestingly, we found that fluoride is a noncompetitive inhibitor of SGAP, in contrast to what was published previously [47], suggesting that the water ⁄ hydroxide molecule is bound similarly in the free enzyme and in the enzyme–substrate complex. Results pH-dependent activity profile The proposed catalytic mechanism for SGAP involves a zinc-bound water ⁄ hydroxide as a nucleophile (Fig. 1). Indeed, the crystal structures of SGAP dem- onstrate that such a water molecule bridges between the two active site zinc ions in an appropriate position, where it acts as a nucleophile in the first stage of the catalytic reaction [26,27]. To verify that the nucleophile is generated from the zinc-bound water molecule, we determined the pH dependence of k cat for the hydroly- sis of leucine-para-nitroanilide (Leu-pNA) under satur- ating substrate concentrations (4 mm) at 298, 303 and 308 °K (Fig. 2). At all three temperatures at pH values below 7.0, logk cat was found to be strongly dependent on the pH, providing slopes of 1.1–1.3. This behavior (slopes of ± 1) is typical of monobasic acids and indi- cates that a single ionization step controls the reaction rate [50]. At pH values above 7.0, logk cat was less affected by the pH. The point of intersection of the two regions is the kinetic pK a of the ionizing groups on the ES complex [51]. As the proton dissociation constant is a thermodynamic parameter, a change in temperature can result in alteration of the pH activity curve. The pK a at each temperature was determined and plotted against the inverse absolute temperature (Fig. 3). From the pK a versus the 1 ⁄ T plot, the enthalpy of ionization (DH ion ) could be obtained, resulting in a value of 30 ± 5 kJÆmol )1 . This enthalpy of ionization value is typical of a zinc-bound water molecule [52]. Thus, the k cat dependence on the pH could reflect the ionization of the zinc-bound water to hydroxide. Inhibition of SGAP by fluoride and phosphate ions Based on the crystal structures of native SGAP, the metal center in the active site binds a water molecule (or a hydroxide ion), which bridges almost symmetri- cally between the two zinc ions [26–28]. To verify the nature of the metal–water ⁄ hydroxide binding and to determine whether one or both metal ions act as Lewis acids in catalysis, we investigated the inhibition of Mechanism of an aminopeptidase from S. griseus Y. F. Hershcovitz et al. 3866 FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS SGAP by fluoride and phosphate anions. Anions such as fluoride and phosphate have been widely used to probe the binding of water ⁄ hydroxide to metal ions in the active site of metalloenzymes [53–58]. Inhibition of SGAP by fluoride and phosphate anions was investi- gated by determining the initial rates of the hydrolysis of Leu-pNA as a function of the inhibitor concentra- tion (0–80 mm NaF or 0–50 mm NaH 2 PO 4 ) at several substrate concentrations (0.1–10 mm). For both anions, the resulting data were found to fit best to a noncom- petitive mode of inhibition (Figs 4 and 5) [59]. In this mode of inhibition, the inhibitor and the substrate (Leu-pNA in this case) bind independently at different sites, namely, the inhibitor binds equally well to the free enzyme or to the enzyme–substrate complex, and A B C Fig. 2. pH dependence of the observed k cat of Leu-pNA hydrolysis by SGAP at different temperatures. (A) 25 °C; (B) 30 °C; (C) 35 °C. The plot used to estimate the pK a at each temperature. Fig. 3. Plot of pK a versus the inverse temperature for the hydro- lysis of Leu-pNA. The enthalpy of ionization, DH ion ¼ 30 kJÆmol )1 , was calculated from the slope of the line. A B Fig. 4. Inhibition of SGAP by fluoride. (A) A representative plot of the Lineweaver–Burk plot for determination of the mode of inhibi- tion at various fluoride concentrations at pH 8. The plots fit the non- competitive inhibition mode. The reaction solution contained 50 m M Mops, 20 lM ZnCl 2 and 1 mM CaCl 2 . Fluoride concentrations were 0.0 (j), 10 (h), 20 (d), 50 (s) and 80 (m)m M NaF. (B) Dixon plot for determination of noncompetitive inhibition. Y. F. Hershcovitz et al. Mechanism of an aminopeptidase from S. griseus FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS 3867 the substrate binds equally well to the free enzyme or to the enzyme–inhibitor complex [42,60]. For purely noncompetitive inhibition, a Dixon plot of 1 ⁄ V versus the inhibitor concentration is expected to yield a straight line for a given substrate concentration (Figs 4B and 5B) [61]. Similar experiments with NaCl instead of NaF or NaH 2 PO 4 ÆH 2 O resulted in no inhibi- tion up to concentrations of 0.8 m NaCl at pH 8, indi- cating that the reaction is not influenced by ionic strength (at the tested concentrations) and, as expected, the binding of Cl – to hard acids is much smaller than that of F – [62]. Such a binding difference was also reported for AAP [35] and is also expected for the zinc ions of SGAP, which are situated in a generally positive environment and hence behave as relatively hard Lewis acids. To further confirm the displacement of the hydroxide nucleophile by the fluoride anion, the pH dependence of the pK i was determined. The purely noncompetitive behavior of fluoride towards SGAP was exhibited over a pH range of 5.9–8.0. However, the pK i value remained constant at low pHs and decreased at pH values above 7.0 (Fig. 6). The point of intersec- tion of the two linear regions corresponded to pH 7.0. These data fit a mechanism involving a deprotonation step from a water molecule to produce a hydroxide ion under conditions in which, at pH values > 7.0, the fluoride ion (the inhibitor) can be replaced by a coordi- nated water ⁄ hydroxide bound to the two zinc ions in a noncompetitive mode [51,60]. Solvent isotope effect The proposed catalytic mechanism of SGAP involves two proton transfers, suggesting that the reaction rate could be affected by solvent isotope effects, typical of catalytic mechanisms involving general acids or general bases. The magnitude of the solvent isotope effect depends of course on the rate-limiting step in the reac- tion, which could include the protonation or deproto- nation steps and ⁄ or the generation of the nucleophile and the collapse of the tetrahedral intermediate (Fig. 1) [63]. To study the protonation events via the catalytic pathway, and to confirm the role of Glu131 as a proton shuttle in catalysis, we carried out the reaction in the presence of D 2 O. The k cat values for both SGAP and the catalytic mutant, E131D, were measured at different D 2 O ⁄ H 2 O ratios at pH values of 6.5 and 8.0. Data were plotted as the rate ratio V n ⁄ V 1 versus the atom fraction of deuterium (n), where V n corresponds to the k cat value obtained at a particular fraction of deuterium (n), and V 1 corresponds to the k cat value in 100% D 2 O (Fig. 7). Interestingly, the presence of D 2 O in solution reduced the catalytic A B Fig. 5. Inhibition of SGAP by phosphate ion. (A) A representative plot of a Lineweaver–Burk plot for determination of the mode of inhibition at various phosphate ion concentrations (Na 2 H 2 PO 4 ÆH 2 O) at pH 7.2. The plots fit noncompetitive inhibition mode. The reac- tion solution contained 50 m M Mops, 20 lM ZnCl 2 and 1 mM CaCl 2 . Fluoride concentrations were 0.0 (j), 10 (h), 20 (d), 30 (s), 40 (m) and 50 (n )m M Na 2 H 2 PO 4 ÆH 2 O. (B) Dixon plot for determination of noncompetitive inhibition. Fig. 6. pH dependence of the fluoride ion inhibition Michaelis con- stant (K i ) for Leu-pNA hydrolysis by SGAP. The pK i at each tem- perature was calculated from the data of initial velocities at different substrate and NaF concentrations using GraFit, version 5.0 for noncompetitive inhibition. Mechanism of an aminopeptidase from S. griseus Y. F. Hershcovitz et al. 3868 FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS activity for both SGAP and the catalytic mutant E131D, resulting in solvent isotope effects of 1.67 and 2.52, respectively, at pH 8; and 2.10 and 2.92, respect- ively, at pH 6.5 (Table 1). The profound solvent iso- tope effect indicates that a proton transfer is involved in the rate-limiting step of the reaction [64]. At pH 8.0, for both SGAP and E131D, there was a linear correla- tion between the rate ratio (V n ⁄ V 1 ) and the atom frac- tion of deuterium (n), suggesting the involvement of a single protonation step in the catalytic reaction at this pH (Fig. 7A,C). However, at pH 6.5, the relation between the rate ratio and the atom fraction of deuter- ium, for both SGAP and E131D, fitted best to a poly- nomial function. This suggests that, at pH 6.5, at least two proton transfers are involved in the rate-limiting steps of the reaction (Fig. 7B,D). To further analyze the number of proton transfers in catalysis, the c method of Albery [65] was applied. This method is based on the observation that the maximum deviation between theoretical proton-inventory curves V n (n) for different mechanistic models occurs at the midpoint of the isotopic solvent mixture (V m , n ¼ 0.5). Thus, it is best to compare various models with the observed midpoint solvent isotope effect, V m ⁄ V 1 . Equations 1–3, derived by Elrod et al. [65] were accordingly used to calculate the predicted values of V m ⁄ V 1 for three gen- eral models. One proton catalysis: V m V 1 ¼ð1 À n m Þ V 0 V 1  þ n m ð1Þ Two-proton catalysis (equal isotope effects): V m V 1 ¼ð1 À n m Þ V 0 V 1  1 2 þ n m "# 2 ð2Þ Generalized solvation changes: V m V 1 ¼ V 0 V 1  ð1Àn m Þ ð3Þ At pH 8.0, the observed values, for both SGAP and its catalytic mutant, E131D, fitted best the model of a AC BD Fig. 7. Rate ratio (V n ⁄ V 1 ) as a function of atom fraction of deuterium (n) for SGAP and its mutant E131D. V n is the k cat value obtained at a particular fraction of deuterium (n), whereas V 1 is k cat in 100% deuterium oxide. (A) SGAP pH 8.0; (B) SGAP pH 6.5; (C) E131D pH 8.0; (D) E131D pH 6.5. The activity was determined in Mops buffer at the appropriate pH, in 20 l M ZnCl 2 ,1mM CaCl 2 and 4 mM Leu-pNA in different ratios of D 2 O ⁄ H 2 O. At pH 8.0 for SGAP and E131D, the data fitted a linear regression curve that describes a one-proton transfer solvent isotope effect. At pH 6.5, a polyno- mial function was fitted for both, describing at least a two-proton transfer solvent iso- tope effect. Table 1. Experimental versus calculated midpoint solvent isotope for the hydrolysis of Leu-pNA by SGAP and its E131D catalytic mutant. Enzyme V 0 ⁄ V 1 Midpoint solvent isotope effect V m ⁄ V 1 Calculated midpoint solvent isotope effect One proton Two protons Generalized solvations changes SGAP pH 6.5 2.10 1.43 1.55 1.50 1.45 E131D pH 6.5 2.92 1.78 1.95 1.83 1.70 SGAP pH 8.0 1.67 1.35 1.34 1.31 1.29 E131D pH 8.0 2.52 1.83 1.76 1.67 1.59 Y. F. Hershcovitz et al. Mechanism of an aminopeptidase from S. griseus FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS 3869 single proton transfer in catalysis [(Eqn (1)] At pH 6.5, the values fitted best a model with two proton trans- fers; however, they could also be fitted to a model involving general solvation changes [Eqns (2) and (3)]. Thus, using two different data analysis approaches, the solvent isotope effects observed for SGAP at pH 6.5 indicate that there are at least two proton transfers in the catalytic pathway and that at this pH these proton transfer steps limit the hydrolysis of the substrate (Table 1). Temperature dependence of k cat and K m To verify the exact role of Glu131, either in binding or catalysis, the kinetic parameters (K m , k cat ) were meas- ured at temperatures between 293 and 329 °K for both SGAP and its catalytic mutant E131D (Fig. 8). We previously verified by differential scanning calorimetry and activity measurements that the melting tempera- ture of SGAP is 348 °K, and that both the native and the mutant enzymes are completely active and stable (at least for 20 min) at 329 °K. In principle, with a rapid equilibrium mechanism (K m ¼ K d ) (dissociation constant, k -1 ⁄ k 1 ), the kinetic constant, K m , usually cor- responds to the formation of the enzyme–substrate complex, E + S fi (ES), whereas k cat characterizes the bond breaking and ⁄ or making step during the formation of the transition state, ES fi (ESÆÆEP)à. Enzyme–substrate interaction E+Sfi (ES) For rapid equilibrium systems where K m ¼ K d , a plot of ln(1 ⁄ K m ) versus 1 ⁄ T provides the standard enthalpy change (DH°) for the enzyme–substrate binding reac- tion, E+Sfi (ES) (Fig. 8A,C). The free energy value (DG°) for the binding can be calculated from the standard free energy equation, DG° ¼ –RTln1 ⁄ K m , and the corresponding entropy (DS°), can be extracted from the standard Gibbs relationship, DG° ¼ DH° ) TDS°. Using these simple definitions, we could calculate the main thermodynamic parameters, free energy, enthalpy and entropy for the reaction catalyzed by SGAP (Table 2). These parameters, as calculated for the step involving the enzyme–substrate interaction, appeared to be quite similar for SGAP and its catalytic AB CD Fig. 8. Temperature dependence of the kinetic parameters for SGAP hydrolysis of Leu-pNA at pH 8. (A,C) Temperature dependence of 1 ⁄ K m in SGAP and E131D, respectively. (B,D) Arrhenius plot: tempera- ture dependence of k cat in SGAP and E131D, respectively. The plots were used to determine the thermodynamic parameters of the SGAP reaction steps. Table 2. Thermodynamic parameters for the hydrolysis of Leu-pNA by SGAP and its E131D mutant. Reaction step SGAP E131D Enzyme–substrate interaction DG° (kJÆmol )1 ) )2 )1.5 E+S fi (ES) DH° (kJÆmol )1 ) )39 )38 DS° (J ⁄ mol*K) )122 )121 Formation of the transition state DGà (kJÆmol )1 ) +59 +81 ES fi (ESÆÆEP)à DHà (kJÆmol )1 ) +29 +38 DSà (J ⁄ mol*K) )100 )144 E a (kJÆmol )1 )3241 Mechanism of an aminopeptidase from S. griseus Y. F. Hershcovitz et al. 3870 FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS mutant E131D. For example, at 303 °K the K m values were 0.45 and 0.58 mm, for SGAP and E131D, respectively. Hence, the replacement of Glu131 by Asp did not significantly affect the initial binding interac- tion of the substrate with the enzyme. Attaining the transition state ES fi (ESÆÆEP)à As described above, k cat is directly correlated with the generation rate of the transition state (ESÆÆEP)à.A plot of log V max versus 1 ⁄ T provides the activation energy (E a ) for the step involving the generation of the transition state. The first-order rate constant, k cat ,ina simple rapid equilibrium reaction refers to V max ⁄ [E], where the enzyme concentration does not change throughout the experiment [66]. Thus, an Arrhenius plot of lnk cat versus 1⁄ T yields the free activation energy of the reaction [–E a ⁄ R(R ¼ 8.3145 JÆK )1 Æ mol )1 )] (Fig. 8B,D). The other thermodynamic con- stants can be extracted from these data using the equations DGà ¼ –RTln(k cat h ⁄ k B T), DHà ¼ Ea ) RT, DSà ¼ (DHà ) D G à) ⁄ T, where k B , h and R are the Boltzman, Planck and gas constants, respectively. The resulting Arrhenius plot forms a straight line, suggest- ing that the rate-limiting step does not change in the tested range of temperatures (no protein melting) [60]. The calculated activation energies for SGAP and E131D were 32 and 41 kJÆmol )1 , respectively (Table 2). Both values are within the range obtained for typical enzymatic reactions (32–48 kJÆmol )1 ). The replacement of Glu131 by Asp resulted in a significant increase of 9kJÆmol )1 for the activation energy, indicating that Glu131 plays a major role in forming the transition state of the catalytic reaction. Discussion Involvement of a zinc-bound hydroxide as the reaction nucleophile Based on structural studies and ample biochemical evi- dence, the crucial elements in the active site that play an essential role in catalysis are the zinc-bound water ⁄ hydroxide and the carboxylic group of Glu131 [26–28]. From a high-resolution crystal structure of SGAP, it was demonstrated that, in its free native state, a water ⁄ hydroxide molecule is held in position by close interactions with the two active site zinc ions and the acidic side chain of Glu131. To test whether this mole- cule is in fact the active site nucleophile, we determined the enthalpy of ionization (DH ion ) of the hydrolysis of Leu-pNA by SGAP, which was found to be 30 ± 5 kJÆ mol )1 . This value is in the range of the expected ioniza- tion of a zinc-bound water ⁄ hydroxide in solution, DH ion of 20–30 kJÆmol )1 [52]. The enthalpy of ioniza- tion of a carboxylic group is much lower, 5–10 kJÆ mol )1 ; thus, the pK a of the acidic residue is less sensitive to changes in temperature. In calculating DH ion , it is assumed that the deprotonation of the zinc- bound water molecule to the hydroxide nucleophile has a greater effect on the reaction rate than the protona- tion of the peptide bond nitrogen by Glu131. In this regard, the isotope effect studies instead suggest that at pH 8, the protonation of the peptide-bond nitrogen by Glu131 is rate limiting (and not the ionization of the zinc-bound water) (Table 1, Fig. 7). Thus, it is likely that the rate-limiting step does change with pH. How- ever, as can be seen in Fig. 2, the k cat values above pH 7.5 contribute very little to the determined pK a (the point of intersection between the two regions) and therefore the DH ion value is valid. Considering both the crystal structure of the ligand- free SGAP, where a bridging water molecule was found to be bound to the zinc ions of the active site, and the observed DH ion value, it is likely that the zinc- bound water molecule generates the catalytic nucleo- phile of the hydrolytic reaction [26–28,36]. Thus, the primary role of Glu131 is to stabilize the zinc-bound water molecule and to extract a proton from the zinc- bound water. An alternative nucleophile could, in prin- ciple, be the negatively charged carboxylate group of Glu131, as was once suggested for Glu270 of carb- oxypeptidase A [67,68]. In this case, the enthalpy of the reaction should have resembled more the ioniza- tion enthalpy of the acidic residue (5–10 kJÆmol )1 ). Similar enthalpy of ionization results were obtained for other homologous metallopeptidases such as AAP towards the substrate Leu-pNA (25 kJÆmol )1 ) [69], and carboxypeptidase E towards the substrate dansyl-Phe- Ala-Arg (28.9 kJÆmol )1 ) [52]. As expected, for both enzymes, the zinc-bound water ⁄ hydroxide is thought to act as the reaction nucleophile. The binding mode of the water ⁄ hydroxide to the di-zinc center Inhibition of SGAP by fluoride anions was utilized to assess the binding of the water ⁄ hydroxide to the active metal center. Fluoride was found to act as a purely noncompetitive inhibitor of SGAP under all the pH conditions tested (5.9–8.0) with a K i value of 11.4 mm at pH 8.0. A noncompetitive inhibition behavior indi- cates that the inhibitor binds similarly to the free enzyme and to the enzyme–substrate complex [42,61]. As fluoride is likely to replace the bound water, this mode of inhibition suggests that binding of the Y. F. Hershcovitz et al. Mechanism of an aminopeptidase from S. griseus FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS 3871 water ⁄ hydroxide molecule to both zinc ions is the same in the free enzyme as in the enzyme–substrate complex. This notion is further supported by several lines of evidence. In the high-resolution crystal structures of SGAP, the water ⁄ hydroxide molecule is clearly observed in contact with the two zinc ions [26,28,49]. In the structures of SGAP in complex with Met, Leu and Phe, it is evident that each amino acid is bound to the active site through the two oxygens of the carboxy- late group [26,27]. These structures appear to resemble either the transition state (a gem-diolate moiety) or the product of the reaction (the free carboxylate group of the cleaved amino acid residue). In both cases, one of the oxygens (O2), which presumably originated from the substrate carbonyl carbon of the peptide bond, is connected to Zn2, whereas the other oxygen (O1), which presumably originated from the hydroxide nucleophile, is bound to both Zn ions (Zn1 and Zn2) in SGAP [27]. The fact that, in the enzyme–product complex, the coordination number of Zn2 is 5 (His247, Glu132, Asp97 and the two carboxylate oxygens) sug- gests that this coordination number is also maintained in the transition state. Thus, fluoride appears to be replacing a water molecule that is bound to both zinc ions in the transition state. Additional support that the hydroxide nucleophile in the gem-diolate intermediate is stabilized by interac- tions to both metals comes from the structures of SGAP with its reaction products. From these struc- tures, it is evident that the N-terminal amine group of the products is stabilized by three residues, namely, Glu131, Asp160 and the backbone carbonyl group of Arg202, whereas, in the related aminopeptidase AAP from A. proteolytica, the N-terminal amine is in con- tact with one of the zinc ions [26,27]. This mode of binding in SGAP still allows the oxygen atoms of the gem-diolate intermediate to be stabilized by interacting with both metals and Tyr246 [2,26,27]. Thus, the catalytic mechanism of SGAP may not require that the N-terminal of the leaving product will be bound to a single zinc atom, as proposed for AAP [2,14,70]. Further support that the two zinc ions function as Lewis acid-type catalysts comes from comparing the structures of SGAP and blLAP (leucine aminopepti- dase from bovine lens). Interestingly, the latter enzyme utilizes a carbonate ion instead of a carboxylic residue to stabilize the water molecule [34]. The position of this carbonate ion in blLAP corresponds to the posi- tion of Glu131 in SGAP. The crystal structure of blLAP in complex with the transition state analog, l-leucinephosphonic acid, revealed that the two oxy- gens of the phosphate group are bound as a bidentate ligand to one of the zinc ions (Zn1), and one of these oxygens bridges between both Zn ions [33]. Based on this structure, the proposed catalytic mechanism for blLAP indicates that both zinc ions function as Lewis acids and a bridging hydroxide acts as a nucleophile by attacking the substrate carbonyl carbon [33–35]. The importance of both zinc ions for the catalytic activity of SGAP is also supported by previous kinetic studies in which it was demonstrated that a single zinc ion in the catalytic site provides only 50% of activity [39]. Taken together, apparently the water ⁄ hydroxide molecule is bound to both zinc ions in the free enzyme similarly as in the enzyme–substrate complex, provi- ding noncompetitive inhibition by fluoride. A similar mode of inhibition was suggested for other metallo- enzymes such as the purple acid phosphatase from bovine spleen and porcine uterus, in which tetrahedral oxyanions were found to bound in a noncompetitive mode by bridging two iron ions in the active site [55]. Note that Harris and Ming [47] suggested a different mode of SGAP inhibition by fluoride. In their study, fluoride appeared to act as an uncompetitive inhibitor, whereas phosphate ions exhibited noncompetitive inhi- bition, suggesting that fluoride and phosphate ions bind differently [71]. At this stage, we do not have a simple explanation for these contradictory results, other than assuming that they originate from different experimental conditions. In AAP, fluoride was found to act as an uncompetitive inhibitor, suggesting that the hydroxyl nucleophile may be terminally bound fol- lowing substrate binding [35]. Phosphate ions appear to act as noncompetitive inhibitors of SGAP, as was also demonstrated previously by Harris and Ming [47]. However, this result is somewhat puzzling because the phosphate ion is too large to simply replace the water molecule. Indeed, crystal structures of SGAP in com- plex with phosphate reveal that the ion, located in the zinc center, occupies both the space of the water mole- cule and the substrate carbonyl group. Similarly, the location of phosphate was also observed in the human membrane-bound glutamate carboxypeptidase II, in which the Zn[ ]O(phosphate) distances are between 1.75 and 1.93 A ˚ [23]. To explain these results, Harris and Ming suggested that in solution the phosphate ion actually binds at a different location. The number of proton transfers in the reaction The number of proton transfers during the catalytic pathway of SGAP was studied in detail by monitoring the solvent isotope effect on SGAP and its general acid–base mutant E131D, both under different pH con- ditions. At pH 8, the observed isotope effect values were 1.67 and 2.52 for SGAP and the E131D mutant, Mechanism of an aminopeptidase from S. griseus Y. F. Hershcovitz et al. 3872 FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS respectively. Comparison of the observed midpoint- values derived from the rate ratio plots (Fig. 7) to the theoretically calculated values (proton inventory proce- dure) (Table 1) suggests that a single proton transfer is involved in catalysis at pH 8. At this pH, the bridging water molecule is likely to be ionized; thus, the reaction is controlled (rate limiting) by other critical proton transfers in the reaction, the proton transfer from Glu131 (acting here as a general acid) to the nitrogen of the amine leaving group. The isotope effect on E131D was considerably higher than that observed on SGAP at pH 8 (Table 1). This emphasizes the importance of the acidic residue (E131) in facilitating the proton trans- fer to the leaving group at the product generation step of the reaction, and is consistent with the four orders of magnitude decrease in k cat observed for E131D [36]. At pH 6.5, the resulting isotope effect values were 2.1 and 2.9 for SGAP and the E131D mutant, respectively, and the calculated midpoint values for both forms of the enzymes fitted at least two proton transfers in the catalytic pathway (Table 1, Fig. 7). At pH 6.5, the zinc- bound water molecule is less likely to be ionized, and therefore an additional proton transfer is required, resulting in at least two proton transfers in the reaction. Interestingly, the solvent isotope effect observed for E131D was somewhat higher under both pH conditions. This presumably reflects the additional energetic barrier required for catalysis in the catalytic mutant, thus provi- ding further support that Glu131 is involved in both proton transfers. Similar trends in proton transfer were obtained with AAP, in which two proton transfers were observed at pH 6.5 and one proton transfer was observed at the higher pH, for both the wild-type and the corresponding E151D catalytic mutant [37]. Taken together, these results suggest that Glu131 and Glu151 play a similar role in SGAP and AAP, respectively. The role of Glu131 Glu131 in SGAP was previously shown to act as one of the catalytic residues, together with Tyr246 [36]. To ver- ify the specific involvement of Glu131 in binding and ⁄ or catalysis, the kinetic parameters of SGAP and its E131D mutant were determined at several tempera- tures. By knowing the temperature dependence of K m (binding) and k cat (catalysis), it is possible to extract the thermodynamic properties of the main reaction steps (i.e. formation of the activated complex, E+Sfi (EÆÆS)à), and the bond-breaking ⁄ making step, ES fi (ESÆÆEP)à. The measured and calculated thermodynamic parameters of the reaction (i.e. free energy, enthalpy and entropy) for both SGAP and the E131D catalytic mutant were quite similar for the binding step (Table 2). Thus, the E131D replacement appears to affect very little the interaction of the enzyme with its substrate. This is also consistent with the K m values obtained for SGAP and the catalytic mutant [36]. By contrast, the E131D replacement resul- ted in a decrease of four orders of magnitude in k cat , corresponding to an increase of 9 kJÆmol )1 in the acti- vation energy for E131D (Table 2), emphasizing the crucial role of Glu131 in catalysis. These results make sense in terms of the geometry changes involved. For example, shortening the carboxylic side chain by approximately 1.5 A ˚ in the position of the catalytic carboxylic group resulted in a large increase in the acti- vation energy [36]. Interestingly, the transition state entropy, DSà, of E131D, is 44 JÆmol )1Æ K )1 lower than that of SGAP. The activated state can be viewed as an unstable transient phase in which bonds and their orien- tations are disordered [60]. It is possible that, in SGAP, the transition state is characterized by significantly more freedom compared with the catalytic mutant. Conclusions The results of the present study substantiate several catalytic features that characterize the mechanism of action of SGAP. Taking together with the structural data we can state: (a) the catalytic nucleophile is a zinc-bound hydroxide; (b) Glu131 is involved in the deprotonation of the zinc-bound water to form the nucleophilic hydroxide and less involved in substrate binding; and (c) the two zinc ions in the active site par- ticipate in stabilizing the hydroxide nucleophile during catalysis. The overall catalytic mechanism of SGAP appears to be quite similar to the mechanism proposed for AAP. However, the two enzymes differ in several aspects, including the exact role of the two active site zinc ions in catalysis, the detailed sequence of zinc- coordination changes during catalysis and the mode of inhibition of anions such as fluoride and phosphate. Experimental procedures Purification of SGAP The cloning of the SGAP gene, site-directed mutagenesis and the expression and purification of the recombinant pro- teins were performed as previously described [36]. Enzymatic assay The aminopeptidase enzymatic activity was determined at 30 °C in a continuous assay using Leu-pNA (Sigma, St Louis, MO, USA) as a substrate. The reactions were Y. F. Hershcovitz et al. Mechanism of an aminopeptidase from S. griseus FEBS Journal 274 (2007) 3864–3876 ª 2007 The Authors Journal compilation ª 2007 FEBS 3873 [...]... 490–504 Mechanism of an aminopeptidase from S griseus 28 Greenblatt HM, Almog O, Maras B, Spungin-Bialik A, Barra D, Blumberg S & Shoham G (1997) Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.7 5A resolution J Mol Biol 265, 620–636 29 Reiland V, Fundoiano-Hershcovitz Y, Golan G, Gilboa R, Shoham Y & Shoham G (2004) Preliminary crystallographic characterization of BSAP, an extracellular... 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Lipscomb WN (1995) Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography Biochemistry 34, 14792–14800 35 Chen G, Edwards T, D’souza VM & Holz RC (1997) Mechanistic studies on the aminopeptidase from Aeromonas proteolytica: a two-metal ion mechanism for peptide hydrolysis . substantiate several catalytic features that characterize the mechanism of action of SGAP. Taking together with the structural data we can state: (a) the catalytic. Israel Aminopeptidases are exopeptidases that catalyze the removal of N-terminal amino acids from peptides; they are found in bacteria, plants and mammalian