Do N-terminal nucleophile hydrolases indeed have
a single amino acid catalytic center?
Supporting amino acid residues at the active site of penicillin
G acylase
Diana Zhiryakova
1
, Ivaylo Ivanov
2
, Sonya Ilieva
3
, Maya Guncheva
1
, Boris Galunsky
4
and
Nicolina Stambolieva
1
1 Institute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
2 Faculty of Biology, University of Sofia ‘Sv. Kl. Ohridski’, Bulgaria
3 Faculty of Chemistry, University of Sofia ‘Sv. Kl. Ohridski’, Bulgaria
4 Institute of Technical Biocatalysis, Hamburg University of Technology, Germany
The N-terminal nucleophile (Ntn) hydrolase superfam-
ily comprises enzymes sharing a characteristic organi-
zation of the secondary structure in the catalytic
domain, despite the very low sequence homology [1,2].
The reaction mechanism that is suggested to be com-
mon for all Ntn hydrolases resembles that of serine
proteases, involving consecutive enzyme acylation and
deacylation steps. A feature of the catalytic mechanism
Keywords
catalytic mechanism; Hammett plot;

optimized by molecular mechanical modeling and at the AM1 level of the-
ory for three model substrates (with H, a methoxy group or a nitro group
in the para position in the leaving group). Intrinsic interactions of several
functional groups at the active site of PGA are discussed in relation to the
catalytic efficiency of the enzyme. The energy barrier computed for the first
step of acylation (the nucleophilic attack of SerB1) is lower than that for
the second step (the collapse of the tetrahedral intermediate). However, the
electronic properties of the substituent on the leaving group affect the
structure of the second transition state. It is shown that the main chain car-
bonyl group of GlnB23 forms a hydrogen bond with the leaving group
nitrogen, thus influencing the hydrolysis rate. On the basis of our computa-
tions, we propose an interpretation of the complex character of the Ham-
mett plot for the reaction catalyzed by PGA. We suggest a modified
scheme of the catalytic mechanism in which some of the intramolecular
interactions essential for catalysis are included.
Abbreviations
AE, acyl enzyme; AGA, aspartylglucosaminidase; ES, enzyme–substrate; GGT, c-glutamyl transpeptidase; MM, molecular mechanical;
Ntn, N-terminal nucleophile; PGA, penicillin G acylase from Escherichia coli; QM, quantum mechanical; TI, tetrahedral intermediate;
TS, transition state.
FEBS Journal 276 (2009) 2589–2598 ª 2009 The Authors Journal compilation ª 2009 FEBS 2589
of Ntn hydrolases is that the nucleophile (side chain
hydroxyl or thiol group), which attacks the carbonyl
carbon of the scissile amide bond, and the general
base, which accepts the proton from the nucleophile,
belong to the same N-terminal amino acid residue
(Ser, Thr, or Cys). The N-terminal nucleophile is
engaged in a hydrogen bond network, which has stabi-
lizing and activating functions in addition to maintain-
ing the proper spatial structure of the active site [3–5].
Therefore, the catalytic efficiency of Ntn hydrolases

tion of the experimental results, a quantum mechanical
(QM) model of the enzyme active site was constructed.
It illustrates the reorganization of the hydrogen bond
network at the active site, which predetermines the cat-
alytic transformations. Several functional groups in the
proximity of SerB1 were assigned probable roles in
catalysis.
Results and Discussion
The results of the kinetic experiments are presented in
Table 1. All substrates have Michaelis constants of the
same order of magnitude. This confirms an earlier con-
clusion, that the p -substituent on the leaving group
influences the reaction rate by its electronic properties
and does not affect substrate binding (Fig. S1) [15].
On the other hand, the hydrolysis rate varies with the
substituent on the amino moiety of the substrate, con-
firming that the formation of acyl enzyme (AE) is the
rate-limiting step of the reaction. The Hammett plot of
log(k
cat,R
⁄ k
cat,H
) versus r
p
)
is shown in Fig. 1. No cor-
relation was observed between the rate constant and
the van der Waals volume, the Taft steric parameter,
or the hydrophobic parameter of the substituent R.
The Brønsted plots of log( k

slope q = )0.50 ± 0.18. The dependence of the rate
on the electronic factor of the substituent for the
PGA-catalyzed hydrolytic reaction is very distinct as
compared with the other Ntn hydrolases for which
data are available [16,17]. The Hammett plot of the
transpeptidation reaction catalyzed by c-glutamyl
transpeptidase (GGT; EC 2.3.2.2), is biphasic,
displaying a negative slope for the electron-donating
substituents (q = )1.3) and a positive slope for the
electron-withdrawing substituents (q = 0.4). The gly-
cosylasparaginase-catalyzed hydrolysis [aspartylgluco-
saminidase (AGA), EC 3.5.1.26] is also characterized
by a biphasic dependence: substrates with electron-
donating groups give a line with slope q = )0.94, and
substrates with electron-withdrawing groups give a line
with slope q = 0.70. For all three enzymatic reactions
(GGT-catalyzed transpeptidation, and AGA-catalyzed
and PGA-catalyzed hydrolysis), acylation is the rate-
limiting step [16–18]. However, the values of q for
Scheme 1. PGA-catalyzed hydrolysis of a series of phenylacetanilides.
Supporting amino acid residues at PGA active site D. Zhiryakova et al.
2590 FEBS Journal 276 (2009) 2589–2598 ª 2009 The Authors Journal compilation ª 2009 FEBS
PGA are higher than those reported for glycosylaspar-
aginase and GGT, indicating that the PGA-catalyzed
hydrolysis is much more sensitive to the electronic
properties of the substituent on the leaving group. The
third segment, corresponding to strong acceptor
groups on the leaving aniline of the substrate, repre-
sents a substantial difference and a key feature of the
PGA catalytic reaction. It is probably a cumulative

3
N
+
Ar ¡ H
2
NAr + H
+
;pK
a,2
refers to the second dissociation step, H
2
NAr ¡
)
HNAr + H
+
.
PhCH
2
CONHC
6
H
4
-pR
r
p
)
pK
a,1
pK
a,2

CF
3
2-Phenyl-N-[4-(trifluoro-methyl)-phenyl]acetamide 0.65 2.57 24.3
a
17.7 ± 1.6 0.35 ± 0.09 50.6
Br N-(4-Bromophenyl)-2-phenylacetamide 0.25 3.88 26.0
a
7.5 ± 0.9 0.23 ± 0.04 32.6
H N,2-Diphenylacetamide 0.00 4.58 27.0 4.8 ± 0.4 0.14 ± 0.02 34.3
CH
3
N-(4-Methylphenyl)-2-phenylacetamide )0.17 5.07 5.1 ± 0.2 0.11 ± 0.01 46.4
OCH
3
N-(4-Methoxyphenyl)-2-phenylacetamide )0.26 5.30 9.4 ± 0.7 0.10 ± 0.02 94.0
a
For these substrates, pK
a2
values in water were not available. They were calculated from those for dimethylsulfoxide, using a linear correla-
tion [32].
–0.4 –0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
–0.2
0.0
0.2
0.4
0.6
0.8
1
.
0

–
Fig. 1. Hammett plot for the hydrolysis of a series of phenylacetyl
p-substituted anilides catalyzed by PGA. The insert shows the
general formula of the substrates.
Table 2. Interatomic distances in the crystal structures of wild-type PGA and the complexes with phenylacetic acid, penicillin G sulfoxide
(PGSO), and its phenylmethanesulfonyl-serylB1 derivative.
Distance (A
˚
)
Ligand
Protein Data
Bank ID Reference
NaGlnB23
–OcSerB1
OeGlnB23
–NaSerB1
OdAsnB241
–NaSerB1
OaGlnB23
–ligand
2.89 3.18 3.01 1pnk [10]
2.89 3.18 3.01 3.86 (O ⁄ COO
)
) PhCH
2
COOH 1pnl [10]
2.69 2.97 2.89 3.60 (N ⁄ NH) PGSO 1gm9 [11]
2.68 3.26 2.86 3.39 (O ⁄ SO
2
) PhSO

CONHC
6
H
5
in the envi-
ronment of the catalytic and the supporting amino
acids. Table 3 presents the geometry parameters, which
undergo significant changes during catalysis, optimized
by AM1 and HF ⁄ 6-31G** QM computations and
molecular mechanical (MM) modeling with the Dreid-
ing force field. The initial Michaelis complex (ES) fea-
tures spatial approximation of the side chain of SerB1
to the carbonyl carbon of the substrate, which is favor-
able for nucleophilic attack (Table 3 and Fig. S3). The
Michaelis complex of penicillin G with PGA has a
similar structure, and is shown in Fig. S4. In the TS
(TS1), which separates the ES complex from the TI,
the proton transfer from the nucleophile to the general
base is practically completed, and the bond between
Oc of SerB1 and the carbonyl carbon of the substrate
is partially formed. The positive charge of the a-amino
group of SerB1 in TS1 is stabilized by three hydrogen
bonds: two with the side chain carbonyl oxygens of
GlnB23 and AsnB241, and a bifurcate bond with its
own side chain oxygen and the carbonyl oxygen of the
substrate. The hydrogen bond between the negatively
charged Oc SerB1 and the main-chain NH of GlnB23
becomes stronger upon the nucleophilic attack. The
energy of the TI for acetanilide, estimated by AM1
computations, is 29.72 kcalÆmol

a
Distance (A
˚
)
ES
R=H
TS1
R=H
TI
R=H
TI
b
R=H
TI
c
R=H
TS2
R=H
TS2
R=NO
2
AE + H
2
NC
6
H
5
R=H
N M C 1.38 1.43 1.50 1.47 1.45 1.57 (1.57) 1.95 (1.92) 2.87
Oc

SerB1
M Oc
SerB1
2.97 2.80 2.94 2.85 2.86 2.97 (2.96) 2.97 (2.97) 2.94
a
If not designated otherwise, atoms belong to the former substrate.
b
From HF ⁄ 6-31G** QM calculations.
c
From MM calculations with the
Dreiding force field. Optimized geometry parameters without GlnB23 in the model are given in parentheses.
Supporting amino acid residues at PGA active site D. Zhiryakova et al.
2592 FEBS Journal 276 (2009) 2589–2598 ª 2009 The Authors Journal compilation ª 2009 FEBS
restoration of the carbonyl function. The hydrogen
bonds of a-NH
3
+
of SerB1 with OeGlnB23 and
OdAsnB241 are elongated. The protonated general
base is situated closer to the leaving group nitrogen
atom, and the proton transfer for the expulsion of the
aniline is in progress (general acid catalysis).
The untypical Hammett dependence observed for
the PGA-catalyzed hydrolysis of phenylacetyl anilides
indicates that a change in the reaction pathway or the
rate-limiting step occurs depending on the structure of
the substrate. For the model substrate acetanilide, the
energy barrier computed for the first step (the nucleo-
philic attack of SerB1), is 32.34 kcalÆmol
)1

ation, and dominates all other electronic effects on the
reaction center. The model system presented here more
closely reflects the structure and electron density distri-
bution of the real substrates and PGA active site.
In order to interpret the biphasic character of the
Hammett plots available for AGA and GGT, it was
suggested that the breakdown of the tetrahedral struc-
ture proceeded via general acid catalysis, whereby the
proton transfer to the nitrogen of the leaving group
aniline occurs simultaneously with the C–N bond
cleavage [17]. Accordingly, the degree of proton trans-
fer and C–N bond cleavage would both depend on the
electronic nature of the p-substituent [16]. Figure 3
shows the optimized structures of the second TSs of
the PGA-catalyzed hydrolysis of acetanilide (R = H)
and p-nitroacetanilide (R = NO
2
). As can be seen, the
stabilizing interactions of the oxyanion with AsnB241,
AlaB69 and the a-amino group of SerB1 become
weaker in TS2; the carbonyl group of the substrate is
partially restored, and the protonated general base is
properly oriented to give a proton to the leaving group
nitrogen (Table 2). In the case of R = H, the proton
transfer is 40% complete, whereas the amidic C–N
bond is cleaved to a very small degree. The TS resem-
bles the structure denoted TS2a in Fig. 4. The partial
positive charge on the nucleofuge nitrogen is stabilized
by hydrogen bonding with the main chain carbonyl
oxygen of GlnB23. Removing this residue from the

3
) (Fig. 1).
The second TS of acylation for p-nitroacetanilide
features a high degree of C–N bond cleavage and a
very low degree of protonation of the leaving group
(Fig. 4). The aromatic system provides resonance
stabilization of the partial negative charge on the
nitrogen. Electron-withdrawing substituents further
decrease the energy of the stationary point. Starting
from R = H with the increase in the r
p
)
value, TS2
shifts in the direction of TS2b, and the expulsion of an
anilide anion is more favored. The main chain car-
bonyl group of GlnB23 can still be hydrogen-bonded
to the leaving group nitrogen (Fig. 3). In the cases of
R = Br, CF
3
, COOC
2
H
5
or COCH
3
, the resultant
effect is a slow increase of the hydrolysis rate constant
with an increase in r
p
)

3
N H
2
O
Bn
O
H
2
N
O
H
N
+
Bn
R
O
–
H
TS2a
CH
3
N H
3
+
O
Bn
O
H
N
–

hydrogen bonding between the leaving group nitrogen
and the main chain carbonyl of GlnB23 leads to the
formation of a TS similar to TS2b. The subsequent
protonation of the nucleofuge becomes the rate-limit-
ing step of enzyme acylation. This is shown by the
negative slope of the third segment of the Hammett
plot for the strong electron acceptors.
The small difference between the hydrolysis rates of
esters and amides of common acyl moieties is evi-
dence for the effect of GlnB23 at the active site of
PGA on the energetics of the reaction. The values of
k
cat
for methyl 2-phenylacetate and 2-phenylacetamide
are 190 and 50 s
)1
, respectively. For both substrates,
acylation is rate-limiting, as the rate constant of deac-
ylation is over 1000 s
)1
[18]. For comparison, AGA
hydrolyzes the b-methyl ester of Asp faster than the
amide (Asn), the rate constants differing by several
orders of magnitude [21]. The leaving alcohol ⁄ alcox-
ide group cannot form a hydrogen bond with the
main chain carbonyl of GlnB23. Most probably, the
repulsion between the two oxygen atoms destabilizes
the second TS and leads to decreased catalytic effi-
ciency in ester hydrolysis. TyrB444 at the active site
of GGT (Protein Data Bank ID: 2dbx [22]) can inter-

deacyl.
, the increasing negative charge
of the leaving OcSerB1 is balanced by the main chain
NH of GlnB23.
On the basis of the presented kinetic results and the
QM model, we propose a modified scheme of the cata-
lytic mechanism of PGA, in which some of the intra-
molecular interactions essential for catalysis are
included (Scheme 2).
Conclusion
We present an experimental Hammett plot for the
PGA-catalyzed hydrolysis of a series of phenylacetyl
p-substituted anilides. The proposed interpretation of
its complex character is based on an extended QM
model, in which specific ES interactions are taken
into account. Several functional groups in the vicinity
of the catalytic center are assigned functions in catal-
ysis. The a-amino group of SerB1 and the main chain
NH of GlnB23 activate and stabilize the c-hydroxyl
group of SerB1 for nucleophilic attack on the sub-
strate. The protonated general base interacts with the
side chain carbonyl oxygens of GlnB23 and AsnB241,
and contributes to the stabilization of the oxyanion
in the TI. The main chain carbonyl group of GlnB23
forms a hydrogen bond with the leaving group nitro-
gen, thus influencing the hydrolysis rate. The specific
orientation and interaction of several amino acids at
the active site of PGA, combined with the effect of
the substituent on the geometry of the second TS,
leads to a change in the reaction pathway and the

3
, N-methylmorpholine) were mixed in organic solvent
(tetrahydrofuran, chloroform) at 0 °C, and 1.2 equivalents
of phenylacetyl chloride were added dropwise. The mixture
was stirred at room temperature for 1–5 h. At the end of
the reaction, the hydrochloride of the organic base was fil-
tered. When chloroform was used, the reaction mixture was
washed consecutively with 0.1 m HCl, a saturated solution
of NaHCO
3
, and distilled water, and dried over MgSO
4
.
The organic solvent was evaporated, and the residue was
crystallized from ethanol, except for N-(4-cyanophenyl)-2-
phenylacetamide, which was crystallized from water, and
N-(4-nitrophenyl)-2-phenylacetamide, which was crystallized
from benzene. N,2-diphenylacetamide, N-(4-bromophenyl)-
2-phenylacetamide and 2-phenyl-N-[4-(trifluoromethyl)-
phenyl]acetamide were obtained by Schotten–Baumann
acylation: one equivalent of the p-substituted aniline was
dissolved in 10% aqueous NaOH; ethanol was added to
increase the solubility of the reagent. The mixture was
cooled to 0 °C, and 1.2 equivalents of phenylacetyl chloride
were added. The reaction mixture was stirred at room tem-
perature for 1–5 h. The precipitate was filtered, washed
with cold distilled water, and crystallized from EtOH (N,2-
diphenylacetamide was crystallized from methanol ⁄ water).
The purity of the synthesized substrates was confirmed
by means of elemental analysis,

2
CO), 7.34–7.22 (5H, m, C
6
H
4
CH
2
CO), 7.66 (2H,
d, J = 8.5 Hz, NHC
6
H
4
CF
3
), 7.81 (2H, d, J = 8.5 Hz,
NHC
6
H
4
CF
3
), and 10.54 (1H, s, CONH).
Supporting amino acid residues at PGA active site D. Zhiryakova et al.
2596 FEBS Journal 276 (2009) 2589–2598 ª 2009 The Authors Journal compilation ª 2009 FEBS
PGA-catalyzed hydrolysis of the phenylacetyl
p-substituted anilides
Reactions were performed at 25 °C in 0.1 m sodium phos-
phate buffer (pH 7.0) containing 10% dimethylsulfoxide
(v ⁄ v). The total volume of the reaction mixture was
2000 lL. The initial substrate concentration ranged

3
– 260;
Br – 266; H – 260; CH
3
– 265; and OCH
3
– 265. Calibra-
tion curves for both the substrate and the free aniline were
prepared, and initial velocities were calculated, taking into
account both the consumption of the substrate and the lib-
eration of the arylamine with the time. The kinetic experi-
ments with each substrate concentration were performed in
triplicate. The turnover number and the Michaelis constant
were determined by nonlinear regression analysis.
Computational methods
The covalent complex of PGA with the irreversible inhibi-
tor phenylmethanesulfonyl fluoride was taken as an initial
structure (Protein Data Bank ID: 1pnm from the Research
Collaboratory for Structural Bioinformatics Protein Data
Bank, ). It was modified by the follow-
ing procedure, using the ds viewerpro 6.0 software pack-
age (Accelrys Software Inc., San Diego, CA, USA) (now
Discovery Studio: />studio/). The sulfur in the SO
2
group was replaced with a
carbon atom. One of the two oxygen atoms was trans-
formed into O
)
, and the other into nitrogen; thus, the two
double S = O bonds were replaced by C–O

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Supporting information
The following supplementary material is available:
Fig. S1. Putative binding of phenylacetyl p-nitroanilide
at the active site of penicillin G acylase.
Fig. S2. Brønsted plots for the hydrolysis of a series
of phenylacetyl p-substituted anilides catalyzed by
penicillin G acylase from E. coli.
Fig. S3. Michaelis complex of phenylacetanilide with
penicillin G acylase optimized by molecular mechanics
with the Dreiding force field.
Fig. S4. Structure of the Michaelis complex of penicil-
lin G with penicillin G acylase optimized by MM cal-
culations with the Dreiding force field.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Supporting amino acid residues at PGA active site D. Zhiryakova et al.
2598 FEBS Journal 276 (2009) 2589–2598 ª 2009 The Authors Journal compilation ª 2009 FEBS