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Protein Engineering vol.14 no.7 pp.

505512, 2001
The determinants of -amylase pHactivity proles
Jens Erik Nielsen
1,2
, Torben V.Borchert
3
and
Gerrit Vriend
1
1
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1,
69117 Heidelberg, Germany and
3
Department of Enzyme Design,
Novo Nordisk A/S, Novo Alle 1, 2880 Bagsvrd, Denmark
2
To whom correspondence should be addressed. Present address: 4222 Urey
Hall, Department of Chemistry and Biochemistry, University of California,
San Diego, Mail Code 0365, La Jolla, CA 920930365, USA.
Email: jnielsen@mccammon.ucsd.edu
The glycosyl hydrolases present a large family of enzymes
that are of great signicance for industry. Consequently,
there is considerable interest in engineering the enzymes
in this family for optimal performance under a range of
very diverse conditions. Until recently, tailoring glycosyl
hydrolases for specic industrial processes mainly involved
stability engineering, but lately there has also been consider-
able interest in engineering their pHactivity proles. We
mutated four neutral residues (N190, F290, N326 and
Q360) in the chimeric Bacillus Ba2 -amylase to both
charged and neutral amino acids. The results show that
the pHactivity prole of the Ba2 -amylase can be changed
by inserting charged residues close to the active site. The
changes in the pHactivity prole for these neutral
charged mutations do not, however, correlate with the
predictions from calculations of the pK
a
values of the active
site residues. More surprisingly, the neutral neutral
mutations change the pHactivity prole as much as the
neutral charged mutations. From these results, it is
concluded that factors other than electrostatics, presumably
the dynamic aspects of the active site, are important for
the shape of the pHactivity proles of the -amylases.
Keywords: -amylase/electrostatics/pHactivity prole/pK
a
calculations/protein dynamics
Introduction
-Amylases are used in several industrial processes such as
starch liquefaction, laundering, dye removal and feed pre-
processing (Guzman-Maldonado and Paredes-Lopez, 1995).
Several of these processes take place at pH values which are
very different from those where the -amylases perform
optimally (Nielsen and Borchert, 2000) and consequently there
is great interest in changing the pHperformance prole of the
-amylases (Nielsen et al., 1999a; Shaw et al., 1999) and
related enzymes (Fang and Ford, 1998; Wind et al., 1998).
The engineering of pHactivity proles for these enzymes has
proven particularly difcult and in the present paper we present
experiments and calculations that suggest several reasons for
the difculties encountered when engineering the pHactivity
proles of -amylases and related enzymes.
-Amylases
The -amylases consist of three domains called A, B and C.
Domain A is a TIM-barrel [(/)
8
-barrel], which is interrupted
Oxford University Press 505
by an irregular -domain (domain B) inserted between the
third -strand and the third -helix of the TIM-barrel. Domain
C is a Greek key motif which is located approximately on the
opposite side of the TIM-barrel with respect to domain B. The
active site is situated in a cleft at the interface between domains
A and B.
The active site consists of a large number of charged groups,
among which are three acids essential to catalytic activity.
Two of these, Asp231 and Glu261 (numbering according to
the BLA sequence), are believed to be the two catalytic groups.
Asp231 is the catalytic nucleophile, while evidence has been
presented for Glu261 being the catalytic hydrogen donor
(McCarter and Withers, 1996; Uitdehaag et al., 1999). The
third essential acid (Asp328) is believed to assist catalysis by
hydrogen bonding to the substrate and by elevating the pK
a
of
Glu261 (Klein et al., 1992; Knegtel et al., 1995; Strokopytov
et al., 1995; Uitdehaag et al., 1999).
The catalytic reaction is believed to consist of three steps
(Sinnot, 1990; McCarter and Withers, 1994; Davies and
Henrissat, 1995; McCarter and Withers, 1996) (see Figure 1).
Step one is the protonation of the glycosidic oxygen by the
proton donor (Glu261). This is followed by a nucleophilic
attack on the C1 of the sugar residue in position 1 by Asp231
[nomenclature as described by Davies et al. (Davies et al.,
1997)]. After the aglycone part of the substrate has left, a
water molecule is activated, presumably by the now de-
protonated Glu261. This water molecule hydrolyses the
covalent bond between the nucleophile oxygen (of Asp231)
and the C1 of the sugar residue in position 1, thus completing
the catalytic cycle.
-Amylase catalysis is thought to be limited by the protona-
tion of the nucleophile at low pH and by the deprotonation of
the hydrogen donor at high pH (Qian et al., 1994; Strokopytov
et al., 1995; Fang and Ford, 1998). The rate-limiting step at
intermediate pH values is not known. This step has to be largely
independent of pH since many -amylases have a pHactivity
prole with a at top. Binding of the substrate and release of
the product are expected to be independent of pH and it has
therefore been speculated that the rate-limiting step is either
substrate binding or product release at intermediate pH values.
Changing the pHactivity prole
If we accept the assumption that -amylase catalysis is limited
at low pH by protonation of the nucleophile (Asp231) and at
high pH by deprotonation of the hydrogen donor (Glu261), then
the pHactivity prole is determined by the pK
a
values of the
these two active site groups (Kyte, 1995). We consider two types
of pHactivity proles: k
cat
proles and the k
cat
/K
m
proles. The
k
cat
prole of an enzyme is determined by the pK
a
values of
the active site groups in the substrate bound form of the enzyme
(Kyte, 1995).
Since the substrate is present in high concentrations in the
majority of industrial processes where -amylases are used, it
is obviously the k
cat
prole and not the k
cat
/K
m
prole that limits
the activity of the enzyme. In order to change the k
cat
prole, we
J.E.Nielsen, T.V.Borchert and G.Vriend
Fig. 1. Catalytic mechanism of the retaining glycosyl hydrolases.
(I) Protonation of the glycosidic oxygen by the hydrogen donor (Glu261)
and attack on the glucose C1 by the nucleophile (Asp231). Departure of the
reducing end of the substrate. (II) Activation of a water molecule, cleavage
of C1Asp231 covalent bond. (III) Regeneration of the initial protonation
states.
therefore have to identify the factors that determine the pK
a
values of the active site residues when the substrate is bound.
The protein environment certainly is important and although not
much is known about the effect of the substrate, it is undoubtedly
also important, since shifts in -amylase pHactivity proles
have been measured when changing the substrate (Keating et al.,
1998). Also, elegant experiments with Bacillus circulans
xylanase have shown that the pK
a
value of the catalytic
hydrogen donor cycles during the catalytic reaction in order to
full its dual role as hydrogen donor and hydrogen acceptor
(McIntosh et al., 1996).
Changing the substrate is unfortunately not an option when
trying to engineer the pHperformance prole of an -amylase
for a specic industrial process and we are therefore left with
the option of changing the enzyme (in our case by site-directed
mutagenesis) and in that way changing the pK
a
values of the
active site groups and thereby the k
cat
prole.
Changing pK
a
values
The pK
a
value of a residue depends on the free energy difference
between the neutral and the charged states of the residue in the
protein. The free energy difference between these two states is
inuenced both by desolvation effects and by the charges and
dipoles in the protein and the substrate. Desolvation effects and
the interaction with dipoles are short-ranged and therefore result
mainlyfrominteractions withresidues inthe immediate environ-
ment. Mutations that aimat changing the pK
a
value of a titratable
group by changing these energies should therefore be placed in
the vicinity of the titratable group.
Mutations that introduce or remove unit charges can be placed
further away from the titratable group, because the interaction
energy between a titratable group and a unit charge (which may
itself be a part of another titratable group) is less dependent
on distance.
The direction of the expected pK
a
shift when perturbing the
environment of an uncoupled titratable group is summarized in
Figure 2. Using this gure, we can therefore easily predict the
direction of an -amylase pHactivity prole shift, if we assume
that the active site residues of the -amylases are relatively
uncoupled or if the charged residue is placed a short distance
from the active site. Thus, placing a negative charge near the
hydrogen donor will elevate the pK
a
of the hydrogen donor and
give a basic shift in the high-pH limb of the pHactivity prole.
Placing a positive charge close to the hydrogen donor will shift
the basic limb of the pHactivity prole to more acidic values.
Identifying the determinants of -amylase pHactivity proles
Previously (Nielsen et al., 1999a), we constructed 15 mutants in
and around the active site of Bacillus licheniformis -amylase
(BLA), in an attempt to change its pHactivity prole. The
506
mutations in the active site were conservative in nature and were
an attempt to change the pHactivity prole of the enzyme
by changing the hydrogen-bond interactions and the solvent
accessibility of the active site residues. The mutations outside
the active site aimed at changing the pHactivity prole by
introducing or removing a charge and in this way perturbing the
pK
a
values of the active site acids.
The mutations in the active site were found to be highly
deleterious to the activity of the enzyme, whereas most of
the mutations further away fromthe active site did not change the
activity of the enzyme signicantly, but nevertheless they pro-
duced some changes in the pHactivity prole. Unfortunately,
the changes in the pHactivity proles did not correlate with the
predictions from electrostatic potential calculations, prompting
us to suggest that effects other than electrostatics were important
for determining the pHactivity prole.
In this paper, we describe howthese other effects are indeed
important for determining the pHactivity prole. We designed
mutations at four positions around the active site of a chimeric
Bacillus -amylase (Ba2). This enzyme was chosen as a model
system because high-resolution structures are available for
both the apo and holo forms of this enzyme (Brzozowski
et al., 2000). We mutated the original neutral residues at the
four positions to both neutral and charged residues and
examined the effects on the pHactivity prole.
The results indicate that point mutations that are likely to
change the dynamics of the active site can change the pH
activity prole. The effects on the pHactivity prole of a
neutral neutral mutation are so large that effects caused by
neutral charged mutations in this study are also likely to be
dominated by the associated differences in active site dynamics.
Materials and methods
Site-directed mutagenesis
The MegaPrimer method for site-directed mutagenesis
(Sarkar and Sommer, 1990) was used to construct a DNA
fragment carrying the mutation. Mutagenic primers were
designed so as to introduce or remove a unique site in the
gene encoding the hybrid -amylase (Ba2). The mutant DNA
fragments were inserted into a Bacillus expression plasmid in
the context of the Bacillus licheniformis -amylase promoter,
signal sequence and transcriptional terminator. The resulting
mutant plasmids were transformed into Bacillus subtilis.
Mutant colonies were identied by endonuclease digests of
colony polymerase chain reaction (PCR) fragments and con-
rmed by DNA sequencing throughout the region covered by
the mutagenic primer.
Fermentation
Fermentation was carried out at 37C for 5 days in shake
asks containing a complex medium mainly consisting of
potato our, barley our and sucrose soy meal (Bang et al.,
1999; Beier et al., 2000).
Protein purication
The supernatant of the fermentation mixture was isolated by
occulation and centrifugation. During ultraltration the buffer
was changed to 20 mM sodium acetate, pH 5.5. The protein
was subsequently applied to an SP-Sepharose Hi-Load column
(Pharmacia). A dialysis step was used to change the buffer to
20 mM H
3
BO
3
5 mM KCl, pH 9.6. The nal step of the
purication procedure consisted of anion-exchange chromato-
graphy on a Mono-Q Hi-Load column (Pharmacia). All protein
preparations were at least 95% pure as shown by SDSPAGE.
Determinants of -amylase pHactivity proles
Fig. 2. Environmental effects on the pK
a
of titratable residues. The effect of placing a titratable group in a negative, positive and hydrophobic environment is
summarized.
Activity assays
The activity as a function of pH was measured over the
range pH 4.010.5, using the Phadebas -amylase test kit
(Pharmacia). Measurements were carried out in duplicate at
30C in 50 mM BrittonRobinson buffer (50 mM H
3
PO
4

50 mM CH
3
COOH 50 mM H
3
BO
3
) containing 0.1 mM
CaCl
2
. The Phadebas -amylase test kit is based on the release
of blue colour from the substrate (blue-coloured starch) upon
cleavage. Hydrolysis is stopped by adding 1/6 volume of 1 M
NaOH to the reaction mixture. After removal of the unhydro-
lysed blue starch by ltration, the amount of hydrolysed
substrate is proportional to the absorbance at 620 nm.
Since the substrate is insoluble (and added in large quantit-
ies), the activity measurements obtained by this method can
be regarded as k
cat
for insoluble starch.
Stability assays
Stability was measured as the residual activity after incubation
at 30C for 15 min. Measurements were carried out at pH 4.5,
7.0 and 9.0 and were performed in duplicate. None of the
mutants showed any detectable differences from the wild-
type stability.
pK
a
calculations
pK
a
calculations were performed with the WHAT IF pK
a
calculation routines (Nielsen and Vriend, 2001). The routines
apply the hydrogen-bond optimization procedure of Hooft
et al. (Hooft et al., 1996) in order to model each of the protein
protonation states accurately. DelPhi II (Nicholls and Honig,
1991) was used to calculate electrostatic energies. The para-
meters for DelPhi II were set as described previously (Nielsen
507
and Vriend, 2001), namely protein dielectric constant, 16 (for
residues that participate in crystal contacts, have an alternative
accessible rotamer or an average B-factor of 20) or 8 (all
other residues); ionic strength, 0.160 M; solvent probe radius,
1.4 ; and solvent dielectric constant, 80. The calculations
were performed without water molecules, because the inclusion
of water molecules was found to decrease the accuracy of the
pK
a
calculations for a test set of nine proteins (Nielsen and
Vriend, 2001).
A model of Ba2 in complex with malto-nonaose was
constructed from the Ba2 -amylasesacarbose X-ray structure
(Brzozowski et al., 2000). The few changes that were necessary
in order to convert the inhibitor to a substrate were made
using Quanta. The CHARMm 22 parameter set was used as
the source of charges and radii for the substrate in the pK
a
calculations.
Preparation of mutant structures
Mutant structures for use in the pK
a
calculations were designed
using the WHAT IF position-specic rotamer libraries (Chinea
et al., 1995). Mutant structures were inspected visually.
Results
We constructed 12 point mutations to examine the determinants
of the pHactivity prole for Ba2. The activity of all mutant
amylases was within one order of magnitude of the activity
of the wild-type at pH 7.0, with three variants having higher
activity than the wild-type. The stability assays showed that
the pH-dependent stability of the mutants was indistinguishable
from that of the wild-type. The results are summarized in
Table I.
J.E.Nielsen, T.V.Borchert and G.Vriend
Table I. Specic activity of the Ba2 point mutations at pH 7.0 as measured
by the Phadebas assay
Mutant Relative activity (%)
N190D 35.72
N190K 38.0
N190G 38.0
F290E 125
F290K 75.3
F290A 152
N326D 43.4
N326A 49.0
N326L 20.0
Q360E 242
Q360K 61.0
Q360A 70.0
Fig. 3. The position of the point mutations in Ba2. Domain A, green;
domain B, cyan; domain C, light grey. The calcium ions are shown as red
spheres and the sodium ion is shown as an orange sphere. The nonaose
substrate used in the pK
a
calculations is shown in yellow. The active site
acids Asp231, Glu261 and Asp328 are shown in red.
Table II. Distance from mutated residues to the active site acids
Residue/atom Distance to Distance to Distance to
Asp231 C () Glu261 C () Asp328 C ()
Asn190 C 17.5 15.6 17.2
Phe290 C 18.3 13.0 12.4
Asn326 C 13.1 12.6 7.1
Gln360 C 13.2 11.0 11.3
Mutations were clustered at four positions, namely N190,
F290, N326 and Q360 (Figure 3). The sites for the mutations
were chosen reasonably close to the active site in order for
the mutations to have a signicant effect on the pK
a
values of
the active site acids (Table II). Also, we required that the wild-
type residue at the position should be neutral. This was
necessary in order to be able to construct neutral neutral
mutations. Visual inspection and WHAT IFs (Vriend, 1990)
structure analysis module furthermore indicated that mutations
could be made at these positions without perturbing the local
structure signicantly. The apparent k
cat
proles for the wild-
type and the mutants were measured with the Phadebas assay.
These k
cat
proles will be referred to as pHactivity proles
in the following.
N190D/K/G
N190 is positioned in domain B in a turn that points into the
edge of the substrate binding cleft (Figure 3). The residue
508
does not hydrogen-bond to any other residue, except that its
NH
2
group points to Tyr193 and most likely interacts with the
-electrons of the aromatic ring. The rotamer distributions for
Asp and Lys at this position reveal that the Asp and Lys side
chains will most likely point in the same direction as the Asn
side chain, thereby causing almost no changes in the local
structure. The unfavourable interaction of the Asp side chain
with the -electrons of Tyr193 is, however, likely to cause
some rearrangements.
The pHactivity proles of N190D, N190K and N190G
show no signicant changes compared with the wild-type pH
activity prole (Figure 4a).
F290K/A/E
F290 is situated in the -helix that lies between -strand 6
and -helix 6 of the TIM-barrel. This in-between -helix is
not a part of the classical TIM-barrel. Phe290 sits next to
His289, which hydrogen bonds to Ser337. Ser337 is positioned
in the loop that covers the active site and its mutation to
glycine results in a protein with 2% of wild-type activity at
pH 7.0, thus hinting at an important role played by the
hydrogen bond between Ser337 and His289.
The pHactivity proles for F290K, F290E and F290A are
shown in Figure 4b. Both F290E and F290A are more active
than the wild-type and both enzymes are much more active at
basic pH than they would have been if they had had a pH
activity prole shaped as the wild-type protein. F290K is
slightly less active than the wild-type, but its pHactivity
prole has also been shifted slightly to more basic pH values.
N326L/A/D
Asn326 sits in the loop that covers the active site and is fairly
close to Asp328 (Table I). Based on the position-specic
rotamer distributions and a bump analysis, it was judged that
neither a lysine nor an arginine could be accommodated at
this position. Furthermore, the rotamer distribution for leucine
gave only two hits at this position, although visual inspection
suggested that only minor adjustments in the local structure
are needed to accommodate this mutation.
The pHactivity prole of N326A is almost identical with
that of the wild-type (Figure 4c), although the mutant has only
~60% of the wild-type activity at pH 7.0. N326D has relatively
higher activity than the wild-type at basic pH, which is in
agreement with the results reported previously by Takase
(1993).
The pHactivity prole of N326L has lost the characteristic
peak around pH 5.5 and has become almost completely at.
Furthermore, the basic limb of the pHactivity prole is shifted
slightly towards more basic pH values.
Q360K/A/E
The mutants Q360A and Q360K (Figure 4d) have ~50% of
the wild-type activity and their pHactivity proles are very
similar to that of the wild-type. Q360E, on the other hand, is
more than twice as active as the wild-type at pH 6.0 and has
a pHactivity prole which is much more bell-shaped than the
wild-type prole.
pK
a
calculations
pK
a
calculations were carried out as described in the Materials
and methods section. The titration curves for the active site
residues were mostly irregular and did not follow the classical
HendersonHasselbalch shape (Figure 5). It was therefore not
possible to determine a pK
a
value for any of the active site
residues. Instead, we have given a qualitative description of
Determinants of -amylase pHactivity proles
Fig. 4. pHactivity proles for the Ba2 wt and the mutants. (a) wt, N190K, N190G and N190D; (b) wt, F290E, F290K and F290A; (c) wt, N326L, N326A
and N326D; (d) wt, Q360K, Q360A and Q360E.
the differences in the titration curves. The detailed titration
curves for the active site residues in all mutant structures can
be found at http://www.cmbi.kun.nl/gv/nielsen/amylase/pKa/.
Wild-type pK
a
calculations
pK
a
calculations were performed for both the apo and holo
forms of the enzyme. In the apo form, Glu261 and Asp328
form a tightly coupled system that titrates roughly as one
group (hence the large oscillations in the titration curves for
these two residues) with a pK
a
value of ~10, whereas Asp231
titrates with an apparent pK
a
of ~2. This corresponds fairly
well with the established view of the -amylase catalytic
mechanism with Glu261 being the hydrogen donor and Asp231
being the nucleophile. In the calculations with the substrate,
however, the titration curve for Asp231 becomes biphasic with
apparent pK
a
values of ~4 and 7, whereas Glu261 remains
negatively charged over the entire pH range. Asp328 is
predicted to have a pK
a
value of ~8. The calculations for the
holo form thus suggest that Asp328 is the hydrogen donor. It
is, however, not clear how reliable the calculations for the
holo form are as the coordinates for the holo form complex
are derived from an enzymeinhibitor complex rather than
from an enzymesubstrate complex.
pK
a
calculations for the mutants
The pK
a
calculations for the mutants were carried out with
the holo structure of the enzyme to give the pK
a
shifts for the
509
active site acids when the substrate is bound. The perturbations
in the titration curves for the active site residues were fairly
small for all mutations of Asn190 and Phe290. This is in perfect
agreement with the experimental results for the mutations at
position 190, but does not correlate with the results for the
mutations at position 290. Changes were observed for the
mutations of Asn326 and Gln360. These changes are described
in more detail below.
N326D/A/L
The calculations for N326A show an upward shift in the pK
a
value of His327 and a downward shift in the pK
a
value of
Asp328. The calculations for N326L show a slight upward
shift in the pK
a
value of Asp328 and an even smaller upward
shift in the pK
a
value of Glu261. The N326D pK
a
calculations
predict that the pK
a
values of Asp231, His327, Asp328 and
Glu261 increase.
Q306A/E/K
The pK
a
calculations for the mutants in position 360 predict
that the pK
a
value of Asp231 becomes elevated upon all three
mutations. In the case of Q360A and Q360K, the shift is very
large, whereas a smaller shift is seen for Q360E. The pK
a
values of His327 and Asp328 are predicted to become slightly
lower for Q360A. The calculations for Q360K predict that the
pK
a
values for His327 and Asp328 increase.
J.E.Nielsen, T.V.Borchert and G.Vriend
Fig. 5. Calculated titration curves of the three active site acids Asp231, Glu261 and Asp328 in the apo-form and in the holo-form of the enzyme. Asp231,
red; Glu261, magenta; Asp328, green.
Correlation of the calculated pK
a
shifts with the
experimentally observed pHactivity prole shifts
The magnitude and direction of the pHactivity prole shifts
for the Ba2 mutants are not reproduced by the pK
a
calculations.
This is most clearly seen when comparing the calculations
with the experimental results for F290A and N326A. F290A
gives the largest shift in the pHactivity prole, but the pK
a
calculations produce insignicant changes in the active site
pK
a
values for this mutation. In the case of N326A, the pK
a
calculations show a signicant perturbation of the pK
a
values
of Asp328 and His327, but the pHactivity prole for this
mutant is almost identical with that of the wild-type.
The calculations for the mutations that introduce charges
also do not correlate well with the experimental pHactivity
prole shifts and this strongly suggests that long-range
electrostatics are less important for the pHactivity prole of
the -amylases than previously thought.
Discussion
The most pronounced effect on the pHactivity prole for a
neutral neutral mutation is seen for F290A. Phenylalanine
and alanine have different sizes and the large effect is seen
for this mutation is therefore not surprising. The imidazole
ring of His289 packs against the aromatic ring of Phe290 in
the wild-type structure and removing the aromatic ring of Phe
290 (by the F290A mutation) makes His289 more solvent
accessible. The higher solvent accessibility changes the pK
a
of His289 slightly (by 0.1 unit in the pK
a
calculations) and in
this way the F290A mutation changes the electrostatic eld in
the protein. This change in the electric eld cannot, however,
be responsible for the observed change in the pHactivity
prole, since both the F290E and F290K mutations should
change the electrostatic eld even more than F290A. They
should thus produce pHactivity proles that are even more
510
different from the wild-type prole than the pHactivity prole
of F290A. This is, however, clearly not the case and the
changes in the pHactivity prole caused by F290A therefore
have to be ascribed to effects other than electrostatics as
outlined below.
The His289Ser337 hydrogen bond
His289 hydrogen bonds to Ser337 and the change in the pK
a
,
and possibly in the dynamics of His289, is likely to affect the
strength of this hydrogen-bond. Ser337 is situated in a loop
that covers the active site and it is therefore likely that a
change in the Ser337His289 hydrogen bond could affect the
dynamics of the active site. The hydrogen bond between
His289 and Ser337 is known to be important, since the
mutation of Ser337 to glycine produces an enzyme with a
50-fold reduction activity (J.E.Nielsen, G.Vriend and
T.V.Borchert, unpublished results).
The reason for the discrepancy between the pK
a
calculations
and the experiments for the mutants F290K and F290E now
becomes clear, since these mutations are also likely to change
the solvent accessibility and the dynamics of His289. The pH
activity prole shifts resulting from these two mutations are
therefore the combined effects of the charge and the change
in the mobility of His289 that are induced by the mutations.
The pK
a
calculations do not attempt to model such effects and
are therefore unable to reproduce the pHactivity prole shifts.
Asn326
Takase constructed the N326D mutation (Takase, 1993) in the
Bacillus stearothermophilus -amylase and reported the same
downward shift in the pHactivity prole as we found. This
shift is not readily explainable and is possibly due to changes
in the mobility. The neutral neutral mutations in this position
(N326A and N326L) are both likely to inuence the active
site dynamics, but only N326L shows a signicant change in
the pHactivity prole.
Determinants of -amylase pHactivity proles
Fig. 6. An example of the effect of a charged point mutation on a hypothetical GluAsp system. It is seen that the simple predictive rules in Figure 2 break
down when the system of titratable groups is strongly coupled. The electrostatic interaction energies between the titratable groups corresponds approximately
to the calculated interaction energies between Glu261, Asp328 and Lys 234 in Ba2. (a) Schematic representation of the GluAsp system. The pK
a
of each
residue in the absence of the other is 3.5 (intrinsic pK
a
in the Figure). The interaction energy between the two groups is 3.0 ln(10)kT (298.15 K). (b)
Schematic representation of the GluAspLys system. The interaction energy between the Asp and the Glu is the same as in (a). The interaction energy with
the Lys is twice as large for the Asp as for the Glu. (c) The titration curves for the AspGlu system shows that the Asp and the Glu have identical titration
curves. (d) The titration curves for the AspGluLys system shows that the titration curve of the Asp is shifted to more acidic pH values ( lower pK
a
),
whereas the titration curve of the Glu is shifted to more basic pH values ( higher pK
a
), thus defying the rules specied in Figure 2.
Gln360
The mutations at position 360 are remarkable in that Q360E
has a large impact on the pHactivity prole, whereas the
mutations Q360K and Q360A, which are expected to change
the dynamics of the enzyme more than Q360E, have wild-
type pHactivity proles. This suggests that the effect of
Q360E is purely electrostatic, although it is difcult to under-
stand why Q360K does not have an equally large effect on
the pHactivity prole.
Effect of introducing a point mutation on a tightly coupled
system of titratable groups
In constructing the point mutations in BLA and BA2 we have
silently assumed that the effect of inserting a titratable group
near the active site could be predicted using the well-established
rules that are summarized in Figure 2. The pK
a
calculations
for Q360K show that this is not always the case, as the pK
a
of Asp328 is calculated to increase when Gln360 is mutated
to a lysine. This is clearly not what would be expected and
511
shows that counterintuitive effects can indeed be achieved
when a system of tightly coupled titratable groups is perturbed.
The phenomenon is illustrated in Figure 6, where the rules of
Figure 2 are shown to break down for an AspGlu system
much like the two catalytic acids in the -amylases. It is
therefore essential in many cases to use pK
a
calculations to
predict the effect of charged point mutations on the pK
a
values
in the active site.
Conclusion
We have shown that signicant changes in the activity of pH
activity proles of a Bacillus -amylase can indeed be achieved
using site-directed mutagenesis. The shifts in the pHactivity
proles for the mutants did not agree with the calculated
changes in the active site electrostatics.
We speculate that changes in the dynamics of the active site
residues are at least as important for the pHactivity prole
as the changes in the active site electrostatics caused by the
introduction of a charged residue. This is corroborated by
J.E.Nielsen, T.V.Borchert and G.Vriend
the mutations F290A and N326L, which change the pH
activity prole without changing the net charge on the molecule.
This implies that the pK
a
values of the active residues can be
changed signicantly by altering the dynamics of the active
site and thus suggests an alternative approach to the engineering
of pHactivity proles.
A detailed explanation of the effects of the neutral neutral
mutations is difcult, as we do not know the motions that are
important for the catalytic activity of the -amylases. It is
likely that very accurate and long molecular dynamics (MD)
simulations could provide insights into this, but in view of the
present day MD force elds and computer speeds, this is not
a feasible solution. The pK
a
calculation method that we used
does not model heavy atom mobility and the pK
a
shifts induced
by mobility changes can therefore not be reproduced in the
calculations. Furthermore, we may well have underestimated
the value of the dielectric constant in the active site in our
calculations. Employing a higher dielectric constant in the pK
a
calculations would reduce the magnitude of the calculated
shifts in the titration curves, but it is unlikely that it would
give a better qualitative agreement with the experimental data.
The pK
a
calculations also show that the -amylase active
site is a strongly connected system of titratable groups. The
example with a strongly coupled AspGlu system given in
Figure 6 shows that the effect of inserting a titratable group
cannot always be predicted by using the simple scheme of
Figure 2. If the circumstances are right, one can observe the
exact opposite of what was predicted. We speculate that
the shifts in the wrong direction seen for several of the
mutants in this and in the previous study (Nielsen et al.,
1999a) could be partly due to such effects. It seems more
likely, though, that the change in the pHactivity proles result
primarily from the change in the active site dynamics.
The ndings of this study stress the point that dynamics are
an essential part of every enzyme and that rational engineering
of enzyme activity is more likely to succeed if a detailed
description of the enzyme mobility is available when designing
the point mutations.
Acknowledgements
The authors thank Vibeke Holbo for help with the construction and purica-
tion of mutant proteins, Jytte Piil for providing puried wild-type protein
and Rebecca Wade, Barry Honig and Lawrence McIntosh for stimulating
discussions.
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Received September 25, 2000; revised April 24, 2001; accepted May 14, 2001

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