Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

Authors

  • Yifat F. Hershcovitz,

    1.  Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa, Israel
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  • Rotem Gilboa,

    1.  Department of Inorganic Chemistry, The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Israel
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  • Vera Reiland,

    1.  Department of Inorganic Chemistry, The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Israel
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  • Gil Shoham,

    1.  Department of Inorganic Chemistry, The Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Israel
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  • Yuval Shoham

    1.  Department of Biotechnology and Food Engineering and Institute of Catalysis Science and Technology, Technion-Israel Institute of Technology, Haifa, Israel
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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

Abstract

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 kJ·mol−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 noncompetitive inhibitor of SGAP at pH values of 5.9–8 with a Ki 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 pKi experiments resulted in a pKa 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 (Km, kcat) over a temperature 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 resulted in a four orders of magnitude decrease in kcat, corresponding to an increase of 9 kJ·mol−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

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 active site [1–3]. The activity of these enzymes is associated with many central biological processes, such as protein maturation, protein degradation, hormone level regulation, angiogenesis and cell-cycle control [4–8]. Not surprisingly, aminopeptidases play an important role in many pathological conditions, including cancer, cataract, cystic fibrosis and HIV infection. Indeed, antitumor 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 inhibiting 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. 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 glutamate 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 aminopeptidase [(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/general 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 complexes of the enzyme with reaction products were determined [26–28] and used together with biochemical data from SGAP and other double-zinc aminopeptidases [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 intermediate. Presumably, the abstracted proton is transferred 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].

Figure 1.

 The proposed catalytic mechanism of SGAP. An acidic residue (Glu131) activates a zinc-bound water molecule and an additional 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 target peptide bond leading to the formation of a gem-diolate intermediate. The abstracted proton is presumably transferred by the acidic residue (Glu131) to the amine group of the leaving peptide bringing to the breakdown of the intermediate. Dashed lines indicate stabilizing interactions and/or hydrogen bonds in the catalytic pathway; Pep, the incoming peptide/protein substrate.

SGAP and AAP were shown to be quite similar in size, sequence, thermostability and overall structure. Nevertheless, a number of significant features differentiate these apparently homologous enzymes, suggesting 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 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 Zn2+ 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 Ca2+[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. Specifically 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 catalytic 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 proposed 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 demonstrate 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 kcat for the hydrolysis of leucine-para-nitroanilide (Leu-pNA) under saturating substrate concentrations (4 mm) at 298, 303 and 308 °K (Fig. 2). At all three temperatures at pH values below 7.0, logkcat 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 indicates that a single ionization step controls the reaction rate [50]. At pH values above 7.0, logkcat was less affected by the pH. The point of intersection of the two regions is the kinetic pKa 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 pKa at each temperature was determined and plotted against the inverse absolute temperature (Fig. 3). From the pKa versus the 1/T plot, the enthalpy of ionization (ΔHion) could be obtained, resulting in a value of 30 ± 5 kJ·mol−1. This enthalpy of ionization value is typical of a zinc-bound water molecule [52]. Thus, the kcat dependence on the pH could reflect the ionization of the zinc-bound water to hydroxide.

Figure 2.

 pH dependence of the observed kcat of Leu-pNA hydrolysis by SGAP at different temperatures. (A) 25 °C; (B) 30 °C; (C) 35 °C. The plot used to estimate the pKa at each temperature.

Figure 3.

 Plot of pKa versus the inverse temperature for the hydrolysis of Leu-pNA. The enthalpy of ionization, ΔHion = 30 kJ·mol−1, was calculated from the slope of the line.

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 symmetrically 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 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 investigated by determining the initial rates of the hydrolysis of Leu-pNA as a function of the inhibitor concentration (0–80 mm NaF or 0–50 mm NaH2PO4) at several substrate concentrations (0.1–10 mm). For both anions, the resulting data were found to fit best to a noncompetitive 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 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 NaH2PO4·H2O resulted in no inhibition up to concentrations of 0.8 m NaCl at pH 8, indicating 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 pKi was determined. The purely noncompetitive behavior of fluoride towards SGAP was exhibited over a pH range of 5.9–8.0. However, the pKi value remained constant at low pHs and decreased at pH values above 7.0 (Fig. 6). The point of intersection 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 coordinated water/hydroxide bound to the two zinc ions in a noncompetitive mode [51,60].

Figure 4.

 Inhibition of SGAP by fluoride. (A) A representative plot of the Lineweaver–Burk plot for determination of the mode of inhibition at various fluoride concentrations at pH 8. The plots fit the noncompetitive inhibition mode. The reaction solution contained 50 mm Mops, 20 µm ZnCl2 and 1 mm CaCl2. Fluoride concentrations were 0.0 (▪), 10 (□), 20 (•), 50 (○) and 80 (bsl00066) mm NaF. (B) Dixon plot for determination of noncompetitive inhibition.

Figure 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 (Na2H2PO4·H2O) at pH 7.2. The plots fit noncompetitive inhibition mode. The reaction solution contained 50 mm Mops, 20 µm ZnCl2 and 1 mm CaCl2. Fluoride concentrations were 0.0 (▪), 10 (□), 20 (•), 30 (○), 40 (bsl00066) and 50 (bsl00084) mm Na2H2PO4·H2O. (B) Dixon plot for determination of noncompetitive inhibition.

Figure 6.

 pH dependence of the fluoride ion inhibition Michaelis constant (Ki) for Leu-pNA hydrolysis by SGAP. The pKi at each temperature was calculated from the data of initial velocities at different substrate and NaF concentrations using GraFit, version 5.0 for noncompetitive inhibition.

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 reaction, which could include the protonation or deprotonation 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 D2O. The kcat values for both SGAP and the catalytic mutant, E131D, were measured at different D2O/H2O ratios at pH values of 6.5 and 8.0. Data were plotted as the rate ratio Vn/V1 versus the atom fraction of deuterium (n), where Vn corresponds to the kcat value obtained at a particular fraction of deuterium (n), and V1 corresponds to the kcat value in 100% D2O (Fig. 7). Interestingly, the presence of D2O in solution reduced the catalytic 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, respectively, at pH 6.5 (Table 1). The profound solvent isotope 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 correlation between the rate ratio (Vn/V1) and the atom fraction 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 deuterium, for both SGAP and E131D, fitted best to a polynomial 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 γ method of Albery [65] was applied. This method is based on the observation that the maximum deviation between theoretical proton-inventory curves Vn(n) for different mechanistic models occurs at the midpoint of the isotopic solvent mixture (Vm, n = 0.5). Thus, it is best to compare various models with the observed midpoint solvent isotope effect, Vm/V1. Equations 1–3, derived by Elrod et al.[65] were accordingly used to calculate the predicted values of Vm/V1 for three general models.

Figure 7.

 Rate ratio (Vn/V1) as a function of atom fraction of deuterium (n) for SGAP and its mutant E131D. Vn is the kcat value obtained at a particular fraction of deuterium (n), whereas V1 is kcat 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 µm ZnCl2, 1 mm CaCl2 and 4 mm Leu-pNA in different ratios of D2O/H2O. 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 polynomial function was fitted for both, describing at least a two-proton transfer solvent isotope effect.

Table 1.   Experimental versus calculated midpoint solvent isotope for the hydrolysis of Leu-pNA by SGAP and its E131D catalytic mutant.
EnzymeV0/V1Midpoint solvent isotope effect Vm/V1Calculated midpoint solvent isotope effect
One protonTwo protonsGeneralized solvations changes
SGAP pH 6.52.101.431.551.501.45
E131D pH 6.52.921.781.951.831.70
SGAP pH 8.01.671.351.341.311.29
E131D pH 8.02.521.831.761.671.59

One proton catalysis:

image(1)

Two-proton catalysis (equal isotope effects):

image(2)

Generalized solvation changes:

image(3)

At pH 8.0, the observed values, for both SGAP and its catalytic mutant, E131D, fitted best the model of a single proton transfer in catalysis [(Eqn (1)] At pH 6.5, the values fitted best a model with two proton transfers; 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 kcat and Km

To verify the exact role of Glu131, either in binding or catalysis, the kinetic parameters (Km, kcat) were measured 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 temperature 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 (Km = Kd) (dissociation constant, k-1/k1), the kinetic constant, Km, usually corresponds to the formation of the enzyme–substrate complex, E + S→(ES), whereas kcat characterizes the bond breaking and/or making step during the formation of the transition state, ES(ES··EP)‡.

Figure 8.

 Temperature dependence of the kinetic parameters for SGAP hydrolysis of Leu-pNA at pH 8. (A,C) Temperature dependence of 1/Km in SGAP and E131D, respectively. (B,D) Arrhenius plot: temperature dependence of kcat in SGAP and E131D, respectively. The plots were used to determine the thermodynamic parameters of the SGAP reaction steps.

Enzyme–substrate interaction E + S→(ES)

For rapid equilibrium systems where Km = Kd, a plot of ln(1/Km) versus 1/T provides the standard enthalpy change (ΔH°) for the enzyme–substrate binding reaction, E + S(ES) (Fig. 8A,C). The free energy value (ΔG°) for the binding can be calculated from the standard free energy equation, ΔG° = –RTln1/Km, and the corresponding entropy (ΔS°), can be extracted from the standard Gibbs relationship, ΔG° = ΔH° − TΔS°. 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 mutant E131D. For example, at 303 °K the Km 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 interaction of the substrate with the enzyme.

Table 2.   Thermodynamic parameters for the hydrolysis of Leu-pNA by SGAP and its E131D mutant.
Reaction step SGAPE131D
Enzyme–substrate interactionΔG° (kJ·mol−1)−2−1.5
E + S → (ES)ΔH° (kJ·mol−1)−39−38
ΔS° (J/mol*K)−122−121
Formation of the transition stateΔG‡ (kJ·mol−1)+59+81
ES → (ES··EP)ΔH‡ (kJ·mol−1)+29+38
ΔS‡ (J/mol*K)−100−144
Ea (kJ·mol−1)3241

Attaining the transition state ES→(ES··EP)

As described above, kcat is directly correlated with the generation rate of the transition state (ES··EP)‡. A plot of log Vmax versus 1/T provides the activation energy (Ea) for the step involving the generation of the transition state. The first-order rate constant, kcat, in a simple rapid equilibrium reaction refers to Vmax/[E], where the enzyme concentration does not change throughout the experiment [66]. Thus, an Arrhenius plot of lnkcat versus 1/T yields the free activation energy of the reaction [–Ea/R (R = 8.3145 J·K−1·mol−1)] (Fig. 8B,D). The other thermodynamic constants can be extracted from these data using the equations ΔG‡ = –RTln(kcath/kBT), ΔH‡ = Ea − RT, ΔS‡ = (ΔH‡ − ΔG‡)/T, where kB, h and R are the Boltzman, Planck and gas constants, respectively. The resulting Arrhenius plot forms a straight line, suggesting 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 kJ·mol−1, respectively (Table 2). Both values are within the range obtained for typical enzymatic reactions (32–48 kJ·mol−1). The replacement of Glu131 by Asp resulted in a significant increase of 9 kJ·mol−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 evidence, 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 molecule is in fact the active site nucleophile, we determined the enthalpy of ionization (ΔHion) of the hydrolysis of Leu-pNA by SGAP, which was found to be 30 ± 5 kJ·mol−1. This value is in the range of the expected ionization of a zinc-bound water/hydroxide in solution, ΔHion of 20–30 kJ·mol−1[52]. The enthalpy of ionization of a carboxylic group is much lower, 5–10 kJ·mol−1; thus, the pKa of the acidic residue is less sensitive to changes in temperature. In calculating ΔHion, 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 protonation 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. However, as can be seen in Fig. 2, the kcat values above pH 7.5 contribute very little to the determined pKa (the point of intersection between the two regions) and therefore the ΔHion 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 ΔHion value, it is likely that the zinc-bound water molecule generates the catalytic nucleophile 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 principle, be the negatively charged carboxylate group of Glu131, as was once suggested for Glu270 of carboxypeptidase A [67,68]. In this case, the enthalpy of the reaction should have resembled more the ionization enthalpy of the acidic residue (5–10 kJ·mol−1). Similar enthalpy of ionization results were obtained for other homologous metallopeptidases such as AAP towards the substrate Leu-pNA (25 kJ·mol−1) [69], and carboxypeptidase E towards the substrate dansyl-Phe-Ala-Arg (28.9 kJ·mol−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 Ki value of 11.4 mm at pH 8.0. A noncompetitive inhibition behavior indicates 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 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 carboxylate 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) suggests 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 interactions to both metals comes from the structures of SGAP with its reaction products. From these structures, 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 contact 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 aminopeptidase 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 position of Glu131 in SGAP. The crystal structure of blLAP in complex with the transition state analog, l-leucinephosphonic acid, revealed that the two oxygens 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, providing noncompetitive inhibition by fluoride. A similar mode of inhibition was suggested for other metalloenzymes 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 inhibition, 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 following 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 complex with phosphate reveal that the ion, located in the zinc center, occupies both the space of the water molecule 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 Å[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 conditions. At pH 8, the observed isotope effect values were 1.67 and 2.52 for SGAP and the E131D mutant, respectively. Comparison of the observed midpoint-values derived from the rate ratio plots (Fig. 7) to the theoretically calculated values (proton inventory procedure) (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 transfer to the leaving group at the product generation step of the reaction, and is consistent with the four orders of magnitude decrease in kcat 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 providing 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 verify the specific involvement of Glu131 in binding and/or catalysis, the kinetic parameters of SGAP and its E131D mutant were determined at several temperatures. By knowing the temperature dependence of Km (binding) and kcat (catalysis), it is possible to extract the thermodynamic properties of the main reaction steps (i.e. formation of the activated complex, E + S(E··S)‡), and the bond-breaking/making step, ES(ES··EP)‡. 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 Km values obtained for SGAP and the catalytic mutant [36]. By contrast, the E131D replacement resulted in a decrease of four orders of magnitude in kcat, corresponding to an increase of 9 kJ·mol−1 in the activation 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 Å in the position of the catalytic carboxylic group resulted in a large increase in the activation energy [36]. Interestingly, the transition state entropy, ΔS‡, of E131D, is 44 J·mol−1·K−1 lower than that of SGAP. The activated state can be viewed as an unstable transient phase in which bonds and their orientations 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 participate 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 proteins 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 carried out directly in 1-mL cuvettes, positioned in a temperature-controlled cuvette-holder hooked to a regulated water-bath. A termocouple sensor was placed inside the cuvette to verify the exact temperature in the cuvette. The assay solution (650 µL total volume) contained 50 mm Mops, pH 8, 1 mm CaCl2 (SGAP was shown to be activated by Ca2+ ions) [10,46] and 0.02 mm ZnCl2, which were mixed together with the appropriate diluted enzyme and substrate concentrations in the range 0.1–10 Km. After the reaction was initiated by adding the substrate, the increase in absorbance at 405 nm was monitored continuously using an Ultrospec 2100 spectrophotometer (Pharmacia, Uppsala, Sweden). At 405 nm, the extinction coefficient for para-nitroanilide at pH 8, and 30 °C was Δε = 10.2 mm−1·cm−1. The catalytic constants, Km, kcat and Ki were determined by analysis with GraFit, version 5.0 using the appropriate inhibition equations when required [59]. In these experiments the experimental error was ± 5%. The inhibitors NaF and NaH2PO4·H2O were added in concentrations in the range 0–80 mm and 0–50 mm, respectively, and the reactions were initiated by adding together the substrate and the inhibitor. Mops buffer (50 mm) was used for all pHs and the extinction coefficient of leucine-para-nitroanilide was corrected for each of the pH values.

Solvent isotope effect

Enzyme samples [SGAP or SGAP(E131D)] were lyophilized and reconstituted with fresh 99.9% D2O (Sigma). The reaction solution, containing Mops buffer, ZnCl2, CaCl2 and the substrate Leu-pNA (4 mm), was composed with 99.9% D2O. Adjusting the pH of the reaction solution was performed accordingly with NaOD or DCl, both with 99%+ deuterium content (Acros Organics, Geel, Belgium). The kinetic assays were performed at either pH 8.0 or 6.5 at 30 °C. Solutions containing different ratios of H2O/D2O were used to determine the kcat values. These solutions were prepared by diluting a ten-fold concentrated stock solution of the enzymatic solution in D2O with the appropriate amounts of D2O and H2O.

Acknowledgements

This study was supported by the Otto Meyerhof Minerva Center for Biotechnology, Technion, established by the Minerva Foundation (Munich, Germany). Y. S. holds the Erwin and Rosl Pollak Chair in Biotechnology.

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