Human salivary a-amylase Trp58 situated at subsite )2 is critical
for enzyme activity
Narayanan Ramasubbu
1
, Chandran Ragunath
1
, Prasunkumar J. Mishra
1
, Leonard M. Thomas
2
,
Gyo¨ ngyi Gye
´
ma
´
nt
3
and Lili Kandra
3
1
Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA;
2
Howard Hughes Medical
Institute, Division of Biology, California Institute of Technology, Pasadena, CA, USA;
3
Department of Biochemistry,
Faculty of Sciences, University of Debrecen, Hungary
The nonreducing end of the substrate-binding site of human
salivary a-amylase contains two residues Trp58 and Trp59,
which belong to b2–a2 loop of the catalytic (b/a)
8

Keywords: salivary a-amylase; site-directed mutagenesis;
subsite engineering; oligosaccharide hydrolysis; crystal
structure.
a-Amylases (a-1,4-
D
-glucan glucanohydrolases, EC 3.2.1.1)
are endoglucanases, widely distributed in all three domains
of life (Bacteria, Archaea and Eucarya), and catalyze
reactions such as hydrolysis and transglycosylation of
polysaccharides [1,2]. These enzymes, belonging to the
glycoside hydrolase family 13 [3], possess very low overall
sequence similarity among the various members; nonethe-
less, in four small regions around the active site, the
members exhibit a strong sequence similarity [4–6] and
harbor the (b/a)
8
barrel topology [7]. This small number of
conserved but critical short regions whose residues are lined
up along the surface of a deep cleft carries out substrate
binding and catalysis in a-amylases [2].
In humans, a-amylase is present in both salivary and
pancreatic secretions; the overall primary sequences of the
pancreatic and salivary a-amylases are highly homologous,
and exhibit a high level of structural similarity [8,9]. Human
salivary a-amylase (HSAmy) is monomeric, calcium binding
protein with a single polypeptide chain of 496 amino acids
[9]. The structure of HSAmy consists of three domains:
domain A (residues 1–99, 170–404), domain B (residues
100–169) and domain C (residues 405–496). The domain A
adopts a (b/a)

MPD, 2-methyl-2,4-pentanediol; PNP, p-nitrophenyl.
Enzyme: a-amylase (a-1,4-
D
-glucan glucanohydrolase) (EC 3.2.1.1).
(Received 9 March 2004, accepted 23 April 2004)
Eur. J. Biochem. 271, 2517–2529 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04182.x
Subsite mapping of the substrate-binding site has also
been reported based on the crystal structure of HSAmy in
complex with a pseudohexasaccharide inhibitor derived
from acarbose [13]. This structure has provided the detailed
stacking and hydrogen bond interactions occurring at
subsites )4 through +2. The glucose moieties occupying
the subsites )1 through )4 are each involved in a number
of interactions with the protein atoms. While subsite )1
interacts with domain A (Arg195, Asp197, Glu233, His299
and Asp300) and domain B residues (His101 and Leu165),
the subsite )4 interacts only with domain B residues
(Asn105, Asp147 and Ser163). These residues are dispersed
in the loops following the strands b2 through b7. In contrast,
subsites )2and)3 interact with residues Trp58, Trp59 and
Gln63 (contained in a loop connecting b2anda2) and
His305 (in mobile loop 304–310). Although the residues
Trp58 and Trp59 are present in a number of a-amylases of
the Eukarya family, there are a few enzymes with Ala at
position 58 and a Tyr at 59 ([18]; follow the links Multi-
alignments and then Eukaryota at mica.
urv.es/pujadas/AAMY/AAMY_01/).
The two aromatic residues, Trp58 and Trp59 interact
with the bound substrate to different extent [8–13]. The
residue Trp59 is involved in a stacking with the 4-amino-4,

nucleotide sequences used in this study are given below.
Sequencing was performed at the DNA Sequencing
Resource Center at the Rockefeller University, New York.
Bacterial strain, media and plasmids
Bac-To-Bac Baculovirus Expression System was used to
generate recombinant and mutant proteins using procedures
outlined previously [19,20]. The following forward pri-
mers (5¢-CCTTTCAGACCTXXXTGGGAAAGATAC-
3¢, where XXX ¼ GCG, CTG, and TAC, respectively, for
W58A, W58L and W58Y) and the corresponding reverse
oligonucleotide primers used to create the mutants studied
in this paper. For W59A and W59L, the forward primer
was designed based on W58 mutation except for
the position change (5¢-CCTTTCAGACCTTGGXXXGA
AAGATAC-3¢,whereXXX ¼ GCG, CTG, respectively,
for W59A, and W59L). All primers were used in vector
pFASTBAC1 (Invitrogen) into which HSAmy gene was
cloned [19]. The mutations were verified by nucleotide
sequencing of the HSAmy cDNA using appropriate primer.
The plasmid pFASTBAC1 with mutant HSAmy was used
to transform into MAX EFFICIENCY DH10BAC
TM
Fig. 1. Conformational space occupied by Trp58 in wild-type HSAmy. The Trp58 site of the wild-type HSAmy crystallized with acarbose showing
the interactions involving the Trp residue (PDB Code 1mfv). Note that the side chain of Trp58 enters into a hydrogen bond with the main chain of
Asp356. Note that Asp300 is one of the three catalytic residues. All other contacts are of hydrophobic nature. The distances are given in Angstroms.
All structural figures were drawn using
SETOR
[48].
2518 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004
(Invitrogen) cells that contained baculovirus genomic DNA

Tris/HCl, pH 8.0) containing 2 m
M
CaCl
2
, and centrifuga-
tion, the supernatant was applied to a 3 · 13 cm DEAE-52
cellulose column (Whatman). Bound materials were eluted
from the column as previously described [19]. Fractions
containing recombinant protein were pooled based on
SDS/PAGE [21] and Western blotting and dialyzed against
cold deionized water using Spectra/Por2 (MWCO of
12–14 000 Da; Spectrum Medical Industries, Inc.) and
lyophilized. At this stage, the enriched enzymes were
subjected to a BioGel P60 size exclusion chromatography
following a procedure described previously [22]. After
pooling the fractions containing the desired mutant enzymes
based on Western blotting, enzymes with greater than 99%
purity were obtained at approximately 5 mgÆL
)1
of the
culture medium.
The mass and purity of the enzymes were confirmed by
mass spectral analysis using Perspective Biosystems, a DE
Pro MALDI-TOF instrument equipped with a laser at
337 nm and operated with a positive or negative detection
with 6 kV acceleration potential. Samples were analyzed in
delayed extraction linear mode, calibrated externally with
bovine serum albumin (Sigma Chemical Co.). All spectra
were the result of averaging 200 shots.
Enzyme activity assays

M
as determined from molar absorbance at 280 nm
(26.1 for HSAmy) and/or BCA protein assay (Pierce). A
typical reaction was carried out in 100 m
M
HEPES buffer
(pH 7.1) containing 50 m
M
NaCl and 10 m
M
CaCl
2
at
30 °C. All experiments were carried out in triplicate and the
average value is reported.
Hydrolysis of maltooligosaccharides
Assays measuring the products of oligosaccharide hydro-
lysis were carried out using a Varian HPLC (ProStar)
system equipped with a single port manual injector and a
refractive index detector (model number 350). The product
distribution of the hydrolysis of oligosaccharide substrates
by the wild-type and mutant enzymes was determined by
HPLC analyses at a single substrate concentration (0.5 m
M
)
at room temperature. In these experiments, the secondary
attacks on products were avoided by analyzing the reaction
at time points wherein the conversion was < 20%. The
hydrolysates were analyzed using an analytical Dextropak
column (100 · 8 mm) to which a Novapak C18 Guard Pak

Hydrolysis of maltooligosaccharide glycosides
Oligosaccharides labeled with 2-chloro-4-nitrophenyl moi-
ety (CNP) were synthesized from b-cylcodextrin [24].
Incubations of the various CNP-labeled oligosaccharides
in 25 m
M
glycerophosphate buffer (pH 7.0) containing
5m
M
Ca(OAc)
2
and 50 m
M
NaCl were carried out at 37 °C
for 30, 40 and 60 min for W58L. The reactions were
initiated by the addition of enzyme (final concentration of
1.85 n
M
HSA and 18.8 n
M
for the mutant W58L) to the
solution containing 1.0 m
M
of substrate. Samples (20 lL)
were taken at various time intervals and injected into the
chromatographic column. The products were separated on
a Spherisorb ODS2 5 lm column (250 · 4.0 mm) with
acetonitrile–water (13 : 87) as the mobile phase and at a
Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2519
flow rate of 1 mLÆmin

complexes with acarbose, these crystals were soaked with
acarbose (1 m
M
final concentration) in 40% MPD for 24 h
and used for data collection. Diffraction data were collected
on a Mar Research imaging plate area detector system
(W58L) or on a Rigaku R-AXIS IV + image plate area
detector (W58A) using Cu K
a
radiation (1.5418 A
˚
) gener-
ated from a Rigaku RU200 rotating anode generator
operating at 50 kV and 100 mA. The crystals were mounted
on loops (Hampton Research) and flash frozen to )170 °C
in liquid nitrogen. One hundred frames were measured with
a1° oscillation to give 98–100% complete data to 2.0 A
˚
(W58L) or 2.1 A
˚
(W58A). The data frames were exposed
for 10 min each. Intensity data were integrated, scaled and
reduced to structure factor amplitudes using HKL suite of
programs [26]. Data collection statistics are given in Table 1.
The unit cell parameters were found to be isomorphous with
those of the wild-type HSAmy [19].
The refinement of these solutions was carried out using
the CNS package [27] wherein cycles of rigid body
refinement, simulated annealing, positional and thermal B
factor refinements were carried out. Bulk solvent corrections

The refinements were continued by the inclusion of the
sugar atoms. Further examination of the density maps
revealed no additional binding sites in the complex.
The final rounds of refinement were carried out using
maximum likelihood method as implemented in REF-
MAC-5 of the CCP4 package [30]. Solvent molecules were
added using the arp/warp procedure [31] in the CCP4
package. The validity of the water molecules were assessed
on the basis of the presence of a peak at least 3 r in the
difference map, at least one hydrogen bond to a protein
atom (N or O) or if the water molecules were part of a chain
connecting protein atoms, and refinement of thermal factor
less than 50 A
˚
2
. Manual fitting was interspaced between
refinements when necessary. The programs
PROCHECK
[32],
CCP
4and
CNS
were used for model analysis of the final
refined structures. The coordinates and structure factors
have been deposited with the Protein Data Bank [PDB
codes are 1jxj (W58L) and 1nm9 (W58A complex)].
Table 1. Summary of diffraction data collection values and structure refinement statistics. NA, not applicable.
Parameters W58L W58A–acarbose complex
Space group P2
1

˚
2
): protein/solvent/other 17/23/NA 23/29/29
R/R
free
(%)
b
16.6/19.8 15.5/19.3
r.m.s. deviations: bonds (A
˚
)/angles (°) 0.015/1.6 0.009/1.1
a
Last shell: 2.07/2.0 A
˚
; 2.15–2.10 A
˚
.
b
Reflections in the test set (number/%): 1795/5.0; 1564/5.0.
2520 N. Ramasubbu et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Results
Kinetics studies of mutants
Replacements at position 58 were based on decreasing bulk
(Ala and Leu) or partial retention of aromatic character
(Tyr). All mutants gave as a single band in SDS/PAGE after
final purification and no isozyme corresponding to the
glycosylated a-amylase ( 62 kDa) was observed in either
SDS/PAGE or through mass spectral analysis [19]. The
effect of the mutations on the hydrolysis of starch was
examined by comparing the specific activities for starch

m
for
the W58Y mutant, albeit lower than the wild type, is 10-fold
higher than either W58L or W58A suggesting that an
aromatic residue at this position might be necessary. In
sharp contrast, the values for the position 59 mutants were
only approximately twofold lower compared with the wild
typeforstarchaswellasG7-PNPassubstrates.Clearly,the
mutation of Trp58 affects the ground state binding of the
substrate and enzyme activity.
Hydrolysis of maltooligosaccharides
Product distributions were determined by HPLC for the
wild type as well as all three mutants with several
oligosaccharides. A typical chromatogram using G4 is
shown in Fig. 2. The substrates were assayed at 0.5 m
M
,
which was used in the standard assay and in previous studies
[13,20]. For each of the mutants, the sites of cleavage for a
given oligosaccharide and the ratio of the products formed
were determined as described previously [20]. The hydrolysis
of each oligosaccharide at a single concentration was
monitored by means of HPLC with an aid of Dextropak
column. The Dextropak column is able to separate the two
anomers of maltooligosaccharides containing three or more
glucose units. The retention times for the a-andb-anomers
of these oligosaccharides were deduced by first determining
the retention time for the a-anomer using HPLC as
described earlier [20]. Briefly, amylose was used as substrate
under similar conditions and the products were separated by

closely resembling the wild type albeit with significantly
lowered k
cat
/K
m
values (Table 2). In contrast, the other two
Table 2. Parameters for the hydrolysis of starch and oligosaccharides. All assays were performed at pH 7.1. Average kinetic errors in kinetic
parameters: specific activity (± 2–5%) for HSAmy and 15–20% for the mutants; K
m
(± 7–10%) and k
cat
(± 5–7%). N.D., not determined.
Enzyme
Hydrolysis of
x-fold decrease in k
cat
/K
m
for G5-PNP substrate
compared with wild-type
enzyme
Starch
specific activity
(UÆmg of protein
)1
)
Heptasaccharide (G7-PNP) Pentasaccharide (G5-PNP)
k
cat
(s

m
(s
)1
Æm
M
)
HSAmy 66 212 175 0.27 648 131 0.35 372 1
W58A 350 0.43 0.39 1.1 0.60 1.70 0.353 1.09 · 10
3
W58L 356 0.43 0.20 2.1 0.26 1.55 0.168 2.31 · 10
3
W58Y 434 0.80 0.31 2.6 4.1 1.63 2.515 0.15 · 10
3
W59A 38 100 111 0.26 427 N.D. N.D. N.D.
W59L 34 415 75 0.16 468 N.D. N.D. N.D.
H305A
a
5016 12 0.28 43 N.D. N.D. N.D. –
a
[37].
Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2521
mutants, W58L and W58A, lacking the aromatic residue
exhibit apparently altered binding modes.
Hydrolysis of maltooliogosaccharide glycosides
The production of glucose in the hydrolysis by mutants
might have occurred in two different binding modes, where
the subsites from )4 to +1 were occupied or where the
subsites from )1 to +3 were occupied. If the mutation of
the residue Trp58 affected binding at glycone subsites, use of
labeled substrates might provide additional insights into the

affected by the mutation.
The relative rate of formation of each product from the
hydrolysis of a series of oligomeric substrates has been used
to estimate the subsite-binding energy in HSAmy and its
mutants [17]. Using this method the binding affinities for the
four glycone and three aglycone binding sites in the mutant
W58L, with the exception of the two subsites adjacent to the
catalytic site, were calculated using a procedure suggested
by Allen and Thoma [34]. The binding energies for the
subsites )3, )2 and +2 are substantially lower compared
with that of HSAmy (Fig. 3B).
As Trp58 is situated at subsite )2 with the highest affinity
among the glycone binding sites, its mutation affects the
cleavage propensity of individual bonds in maltooligosac-
charides (Fig. 3A; Table 3). Reducing the bulk of the side
chain at position 58 appears to suppress the binding beyond
subsite )2. A reduction of the binding affinities in neigh-
boring subsites is expected for multivalent ligands that bind
in a cooperative manner. Thus, if one binding site shows
reduced affinity, the neighboring binding sites will too,
because the binding sites are not independent of each other.
Because of this reduction in the binding affinity at these
subsites, there is an increase in the productive binding mode
in which glucose from the nonreducing end is generated;
however, this is accompanied by a severe loss in activity
(Table 2).
Structural studies of W58L and W58A
Substitution of a Leu residue for Trp58 causes little
perturbation in the structure (Fig. 4A). The conformation
of the main chain and the orientation of the side chains of

W58L, which do not possess an aromatic residue at position 58, have
distinctly different cleavage pattern than either HSAmy or W58Y. (B)
Subsite maps for HSAmy (solid bar) and W58L (open bar). The arrow
indicates the scissile bond. The reducing end of maltooligomers is
situated at the right hand of the subsite map. Negative energy values
indicate binding between the enzyme and aligned glucopyranosyl res-
idues, while positive values indicate repulsion.
Ó FEBS 2004 Trp58 mutants at subsite )2 of human salivary a-amylase (Eur. J. Biochem. 271) 2523
observed when compared with either W58L or HSAmy
except for an altered orientation of the His305 side chain
compared with HSAmy/acarbose complex (v
2
171°,W58A/
acarbose vs. v
2
)114°, HSAmy/acarbose complex). This
altered conformation alone could not account for the
significant reduction in the k
cat
as a mutation of His305 to
Ala reduced the k
cat
by only 15-fold ([35]; Table 2). While
His305 is known to shift its position in the liganded
structures to interact with the bound oligosaccharide
[11,13], in this structure (W58A complex) part of the loop
(residues 305, 306 and 307) is not well defined. This
suggested that the presence of a well-occupied sugar moiety
at subsite )2 might be required for interaction mobilizing
the entire mobile loop.

for the subsite )1, +1 and +2
atoms). Refinement with partial occupancy for the atoms at
these subsites also did not improve the model. Therefore,
only water molecules, which satisfied the criteria given in the
Materials and methods section, were modeled into the
disordered density.
The structural analyses reveal that the inhibitor binding
at subsites )1, +1 and +2 has little impact on the
interactions and orientation of the catalytic groups in the
active site. The complex W58A/acarbose displayed a
secondary sugar-binding site on its surface centered on the
residues Trp284 (and Tyr276). This binding site has been
previously observed in the complex structures of wild-type
HSAmy and the mutant D306 lacking the mobile loop
residues 306–310 [13]. Smaller oligosaccharides have been
observed to occupy similar surface sites in several
a-amylases including porcine pancreatic a-amylase [36]
and barley a-amylase [37].
Discussion
Enzymatic properties of Trp58 mutants
The major effect of the mutation appears to be the loss of
the catalytic efficiency as illustrated by the decrease in the
k
cat
and an increase in K
m
for smaller oligosaccharides. This
suggested that the transition state stabilization is hampered
by the removal of the bulky side chain in the middle of the
binding pocket and that interactions around subsites )2and

CNP-G
2
CNP-G
3
CNP-G
4
CNP-G
5
CNP-G
6
CNP-G
3
W58L 47 53
HSAmy – –
CNP-G
4
W58L 13 74 13
HSAmy 10 85 5
CNP-G
5
W58L2612710
HSAmy 2 86 12
CNP-G
6
W58L2353028 5
HSAmy 51 44 5
CNP-G
7
W58L1133338132
HSAmy 18 50 30 2

In the study of barley a-amylase, it was shown recently
that Met53 (equivalent to Gln63 at subsite )2in
HSAmy) was required for wild-type kinetic properties
such as affinity [42]. Inadequate binding at subsite )2
caused distortions at the subsite )1. In the mutants
studied here, such a distortion at subsite )1 may not
occur as subsite )1 is fully occupied. The interactions
around this subsite agree well with interactions observed
around subsite )1 in wild-type complex [13]. However,
local rearrangement of some side chain residues around
Trp58 does occur, most notably in His305 and Lys352.
Two water molecules bridging Asp356 and subsite )2
glucose moiety are also absent (Fig. 6). It should be
pointed out, however, that at the present resolution
(2.1 A
˚
), the mobile loop His305 side chain is not well
resolved. This might be taken to be suggestive of the
inability of the loop to become ordered upon saccharide
binding, a characteristic feature in a-amylases containing
such a mobile loop, as critical subsites )2and)3 are not
occupied. Nonetheless, from the structural and kinetic
data obtained in this study for the W58A/L/Y mutation,
it is clear that the residue Trp58 plays a critical role in
the proper binding of the substrates and thus, for
maintenance of the optimal catalytic activity of HSAmy.
The role of Trp58 in enzyme activity
Several conserved residues, dispersed throughout the
sequence, are juxtaposed around the active site of
a-amylases, some of which have been shown to be

link Multialignments). The length of this loop varies in size
in different a-amylases and contains one invariant residue
Tyr62 that provides a stacking interaction at subsite )1. An
examination of the reported a-amylase structures contain-
ing acarbose-derived pseudooligosaccharides revealed that
noncontiguous residues occupy the space occupied by
Trp58-Trp59 in HSAmy. For example, in TAKA-amylase
[46], residues Arg344 and His80 are located at the positions
occupied by Trp58 and Trp59, respectively). Thus, the
stacking interaction provided by the Trp59 in a-amylases
appears to be compensated by His80 in TAKA-amylase.
However, as a result of the variations in the sequence,
HSAmy and TAKA-amylases bind pseudooligosaccharides
in two orientations (Fig. 7) [13,46]. The conformational
freedom of the substrate, if any, in TAKA-amylase is
restricted probably due to the orientation of two peptide
segments TTAYG(72–76) and GDNTV(167–171) around
subsite )3. Modeling studies showed that the residues Trp58
(and Trp59) of HSAmy will encounter severe steric inter-
actions with the pseudooligosaccharide if the sugar units
occupying subsite )2/)3 adopt alternate conformations as
observed in TAKA-amylase (Fig. 7). Interestingly, the size
of the substrate-binding pocket around the glycone subsites
in a-amylase enzymes that possess Trp58Trp59 segment
appears to be larger. Why mammalian a-amylases and some
other bacterial a-amylases possess a larger substrate-binding
pocket is unclear at present. Nonetheless, these amylases
with Trp58-Trp59 segment also possess a loop segment
GHGGA (residues 304–310 in HSAmy and residues 268–
Fig. 5. Difference electron density maps (omit maps) in the mutants W58L and W58A/acarbose complex. (A) Stereodrawing of the 2Fo-Fc omit maps

Second, it might assist the imidazole side chain of His305 to
be properly juxtaposed to interact with the subsite )2
glucose moiety and also assist in ordering the water
structure around these subsites (Fig. 6). Thus, only in the
presence of Trp58, the mobile loop along with His305
repositions to anchor glucose at )2 (and possibly )3)
susbites at the binding pocket. In this regard, Trp58 might
be involved in the pathway in which information flows
between substrate and the catalytic site through His305 [13].
Third, the proximity of the Trp side chain to the catalytic
carboxyl group Asp300 suggests that some distortion of the
negative electrostatic potential might be occurring at the
catalytic site. To test this, atomic charges on the carboxyl
group atoms of Asp300 were evaluated by semiempirical
methods (MOPAC with AM1 as provided in the SYBYL
software, Tripos Inc.). The atomic charges on the atoms
around position 58 in HSAmy, W58L and W58A were
calculated and compared (data not shown). This prelimin-
ary analysis suggested that the overall negative charge on
the two oxygen atoms of the carboxylate group was higher
in the wild type than in the mutants. The positive charge on
the carbon atom of the carboxylate group remained the
same in all three structures. Such distortion of negative
electrostatic potential has been suggested to be partially
responsible for the very low activity when Met53 located at
subsite )2 was replaced either with Tyr or Trp in barley
a-amylase [42]. Whether such an effect occurs in HSAmy is
beyond the scope of this report. Nonetheless, it should be
pointed out that mutation of Asp300 to Asn in human
pancreatic a-amylase, wherein the charge polarization could

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