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-

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,

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

[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)

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

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

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

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)

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

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


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

(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

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
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

, 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-

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

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

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

,
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

2+
ions) [10,46] and 0.02 mm ZnCl
2
, which were
mixed together with the appropriate diluted enzyme and
substrate concentrations in the range 0.1–10 K
m
. 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 De ¼ 10.2 mm
)1
Æcm
)1
.
The catalytic constants, K
m
, k
cat
and K
i
were determined by
analysis with GraFit, version 5.0 using the appropriate inhi-
bition equations when required [59]. In these experiments
the experimental error was ± 5%. The inhibitors NaF and
NaH
2
PO

O
were used to determine the k
cat
values. These solutions were
prepared by diluting a ten-fold concentrated stock solution
of the enzymatic solution in D
2
O with the appropriate
amounts of D
2
O and H
2
O.
Acknowledgements
This study was supported by the Otto Meyerhof
Minerva Center for Biotechnology, Technion, estab-
lished by the Minerva Foundation (Munich, Ger-
many). Y. S. holds the Erwin and Rosl Pollak Chair in
Biotechnology.
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