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

are that: a AAP is almost fully active approximately80% [14,35,43–45], with only one zinc ion in the active site, whereas the corresponding SGAP was shown to be approximately 50% active,

aminopeptidase from Streptomyces griseus

Yifat F Hershcovitz1, Rotem Gilboa2, Vera Reiland2, Gil Shoham2and Yuval Shoham1

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 Kiof 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 experi-ments resulted in a pKavalue 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 tempera-ture range of 293–329K 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 kcat, 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.

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.

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

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 kcatfor 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, logkcatwas 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, 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 (DHion) 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 kcat 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

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 NaH2PO4) 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 kcatof 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 pKa 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 concentrainhibi-tions at pH 8 The plots fit the non-competitive inhibition mode The reaction solution contained 50 m M Mops, 20 l M ZnCl2and 1 m M CaCl2 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.

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 NaH2PO4ÆH2O resulted in no

inhibi-tion up to concentrainhibi-tions 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 pKi was determined The purely

noncompetitive behavior of fluoride towards SGAP was exhibited over a pH range of 5.9–8.0 However, the

pKivalue 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 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

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 (Na2H2PO4ÆH2O)

at pH 7.2 The plots fit noncompetitive inhibition mode The

reac-tion solureac-tion contained 50 m M Mops, 20 l M ZnCl2and 1 m M CaCl2.

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.

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 (Vn⁄ V1) 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 Vn(n) for

different mechanistic models occurs at the midpoint of

the isotopic solvent mixture (Vm, n¼ 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 gen-eral models

One proton catalysis:

Vm

V1

¼ ð1  nmÞ V0

V1

 

Two-proton catalysis (equal isotope effects):

Vm

V1

¼ ð1  nmÞ V0

V1

 1

þ nm

ð2Þ Generalized solvation changes:

Vm

V1

¼ V0

V1

 ð1n m Þ

ð3Þ

At pH 8.0, the observed values, for both SGAP and its catalytic mutant, E131D, fitted best the model of a

Fig 7 Rate ratio (Vn⁄ 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 , 1 m M

CaCl2and 4 m M Leu-pNA in different ratios

of D2O ⁄ 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.

Midpoint solvent isotope effect V m ⁄ V 1

Calculated midpoint solvent isotope effect One proton Two protons Generalized solvations changes

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 kcatand Km

To verify the exact role of Glu131, either in binding or

catalysis, the kinetic parameters (Km, kcat) were

meas-ured at temperatures between 293 and 329K 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 348K, and that both the native and

the mutant enzymes are completely active and stable

(at least for 20 min) at 329K In principle, with a

rapid equilibrium mechanism (Km¼ Kd) (dissociation

constant, k-1⁄ k1), the kinetic constant, Km, usually

cor-responds to the formation of the enzyme–substrate

complex, E + Sfi (ES), whereas kcat characterizes

the bond breaking and⁄ or making step during the

formation of the transition state, ESfi (ESÆÆEP)

Enzyme–substrate interaction E + Sfi (ES) For rapid equilibrium systems where Km¼ Kd, a plot

of ln(1⁄ Km) versus 1⁄ T provides the standard enthalpy change (DH) for the enzyme–substrate binding reac-tion, E + S fi (ES) (Fig 8A,C) The free energy value (DG) for the binding can be calculated from the standard free energy equation, DG ¼ –RTln1 ⁄ Km, 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

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 kcatin 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.

Enzyme–substrate interaction DG (kJÆmol)1) )2 )1.5

DS (J ⁄ mol*K) )122 )121 Formation of the transition state DG (kJÆmol)1) +59 +81

DS (J ⁄ mol*K) )100 )144

Ea(kJÆmol)1) 32 41

mutant E131D For example, at 303K the Kmvalues

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 ESfi (ESÆÆEP)

As described above, kcatis directly correlated with the

generation rate of the transition state (ESÆÆEP) 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 ¼ 8.3145 JÆK)1Æ

mol)1)] (Fig 8B,D) The other thermodynamic

con-stants can be extracted from these data using the

equations DG ¼ –RTln(kcath⁄ kBT DH ¼ Ea) RT,

DS ¼ (DH) DG) ⁄ T, where kB, h and R are the

Boltzman, Planck and gas constants, respectively The

resulting Arrhenius plot forms a straight line,

suggest-ing that the rate-limitsuggest-ing 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

9 kJÆmol)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 (DHion) 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,

DHionof 20–30 kJÆmol)1 [52] The enthalpy of ioniza-tion of a carboxylic group is much lower, 5–10 kJÆ mol)1; thus, the pKa of the acidic residue is less sensitive to changes in temperature In calculating

DHion, 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 kcat values above

pH 7.5 contribute very little to the determined pKa(the point of intersection between the two regions) and therefore the DHionvalue 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 DHionvalue, 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 Ki 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

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,

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 kcatobserved 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

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 + Sfi (EÆÆS)), and the bond-breaking ⁄ making

step, ESfi (ESÆÆEP) 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 resul-ted in a decrease of four orders of magnitude in kcat, 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, DS, of E131D, is 44 JÆmol)1ÆK)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

30C in a continuous assay using Leu-pNA (Sigma, St Louis, MO, USA) as a substrate The reactions were