Báo cáo khoa học: Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334fiGly mutant enzymes pot

Probing the active site of Corynebacterium callunae starch
phosphorylase through the characterization of wild-type
and His334
fi
Gly mutant enzymes
Alexandra Schwarz
1
, Lothar Brecker
2
and Bernd Nidetzky
1
1 Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
2 Institute of Organic Chemistry, University of Vienna, Austria
Glycogen phosphorylases are pyridoxal 5¢-phosphate
(PLP)-dependent glycosyltransferases (EC 2.4.1.1) that
catalyze the reversible phosphorolysis of oligomeric
and polymeric a-1,4-glucan substrates (maltodextrins,
starch, glycogen) [1,2]. The reaction proceeds with
retention of configuration at the anomeric carbon,
yielding a-d-glucose 1-phosphate (Glc1P) as product
in the direction of substrate depolymerization. In spite
Keywords
a-retaining glucosyl transfer; phosphorus
NMR; pyridoxal 5¢-phosphate; saturation
transfer difference NMR; starch
phosphorylase
Correspondence
B. Nidetzky, Institute of Biotechnology and
Biochemical Engineering, Graz University of
Technology, Petersgasse 12, A-8010 Graz,
Austria

Fax: +43 316 873 8434
Tel: +43 316 873 8400
E-mail:
(Received 15 May 2007, revised 1 August
2007, accepted 6 August 2007)
doi:10.1111/j.1742-4658.2007.06030.x
His334 facilitates catalysis by Corynebacterium callunae starch phosphory-
lase through selective stabilization of the transition state of the reaction,
partly derived from a hydrogen bond between its side chain and the C-6
hydroxy group of the glucosyl residue undergoing transfer to and from
phosphate. We have substituted His334 by a Gly and measured the disrup-
tive effects of the site-directed replacement on active site function using
steady-state kinetics and NMR spectroscopic characterization of the cofac-
tor pyridoxal 5¢-phosphate and binding of carbohydrate ligands. Purified
H334G showed 0.05% and 1.3% of wild-type catalytic center activity for
phosphorolysis of maltopentaose (k
catP
¼ 0.033 s
)1
) and substrate binding
affinity in the ternary complex with enzyme bound to phosphate (K
m
¼
280 mm), respectively. The
31
P chemical shift of pyridoxal 5¢-phosphate in
the wild-type was pH-dependent and not perturbed by binding of arsenate.
At pH 7.25, it was not sensitive to the replacement His334 fi Gly. Analysis
of interactions of a-d-glucose 1-phosphate and a-d-xylose 1-phosphate
upon binding to wild-type and H334G phosphorylase, derived from satura-

tion transfer difference NMR experiments, suggested that disruption of
enzyme–substrate interactions in H334G was strictly local, affecting the
protein environment of sugar carbon 6. pH profiles of the phosphorolysis
rate for wild-type and H334G were both bell-shaped, with the broad pH
range of optimum activity in the wild-type (pH 6.5–7.5) being narrowed
and markedly shifted to lower pH values in the mutant (pH 6.5–7.0).
External imidazole partly restored the activity lost in the mutant, without,
however, participating as an alternative nucleophile in the reaction. It
caused displacement of the entire pH profile of H334G by + 0.5 pH units.
A possible role for His334 in the formation of the oxocarbenium ion-like
transition state is suggested, where the hydrogen bond between its side
chain and the 6-hydroxyl polarizes and positions O-6 such that electron
density in the reactive center is enhanced.
Abbreviations
CcStP, Corynebacterium callunae starch phosphorylase; GL,
D-gluconic acid 1,5-lactone; Glc1P, a-D-glucose 1-phosphate; LFER, linear free
energy relationship; PLP, pyridoxal 5¢-phosphate; STD, saturation transfer difference; X1P, a-
D-xylose 1-phosphate.
FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5105
of detailed studies spanning many decades, definite
conclusions about the catalytic mechanism of glycogen
phosphorylases and the exact function of the PLP co-
factor in it are still elusive [2–6]. Figure 1 shows that
an active site His has a central role in the contentious
debate surrounding a putative covalent glucosyl–
enzyme intermediate of a double displacement-like
mechanism of the phosphorylase. The precedent of
sucrose phosphorylase [7–9], mechanistically represent-
ing a large class of retaining glycoside hydrolases and
transglycosidases, would strongly favor some form of

a two-step mechanism, consisting of glucosylation and
deglucosylation of a catalytic group on the enzyme,
typically a carboxylate of Glu or Asp [5,10]. Glycogen
phosphorylase structures reveal that the backbone
amide carbonyl of the His is the only group appropri-
ately placed to function as a nucleophile [4,11–15]
(Fig. 1B). However, despite the vast assortment of
probes used, all searches for a covalent intermediate of
glycogen phosphorylase have proved fruitless so far
[5]. Partly driven by this negative evidence, an alterna-
tive mechanism termed S
N
i-like was proposed, where,
in the direction of phosphorolysis, attack of phosphate
as nucleophile and departure of the oligosaccharide
leaving group occur on the same face of the glucosyl
residue being transferred [2,4–6,11,13]. It involves only
a single transition state that has a highly developed
oxocarbenium ion character. The His is proposed to
stabilize this transition state through electrostatic and
hydrogen bonding interactions of its main chain car-
bonyl and side chain, respectively. The phosphate ion
positioned in a ‘tucked-under’ conformation on the
opposite (a) face of the glucosyl oxoarbenium ion-like
species presumably provides additional electrostatic
stabilization, derived from its interactions with C-1
and O-5 as well as O-2 of the pyranosyl ring
[4,6,11,13] (Fig. 1B). Earlier kinetic studies of wild-type
phosphorylases support this idea by showing coopera-
tive-like (synergistic) binding of phosphate and gluco-

syl oxoarbenium ion mimics such as d-gluconic acid
1,5-lactone (GL) [16,17]. Substitution of His334 in
starch phosphorylase from Corynebacterium callunae
(CcStP) (Fig. 1A) by Gln or Asn caused a substantial
(up to 150-fold) loss in wild-type catalytic efficiency
that was paralleled by a corresponding decrease in
affinity for GL in combination with phosphate, reflect-
ing a change from positive to negative cooperativity in
binding of the two ligands as a result of the site-direc-
ted replacement [18].
In this work, we have substituted His334 with Gly
and analyzed the disruptive effects of the point muta-
tion on active site function of CcStP using steady-state
kinetics and selective NMR probes for the 5¢-phos-
phate group of the cofactor and for bound carbo-
hydrate ligands. The work was carried out to address
three questions in particular, taking into account that,
quite unexpectedly, an H334A mutant of CcStP was
almost as active as the wild-type enzyme [18]. How
does complete removal of the side chain of His334
influence binding and catalysis? Are the properties of
neighboring active site groups, including the PLP
cofactor, affected by the His fi Gly mutation? If suffi-
cient room is vacated in H334G to accommodate
water or another nucleophile in place of the original
methylimidazole group, will this new ligand participate
in the enzymatic reaction such that eventually hydro-
N
O
N

N
O
Pyridoxal
P
O
O
OH
OH
O
OH
OH
OH
O
P
O
O
O
N
O
N
O
N
O
O
N
N
O
O
O
N

O
O
N
O
N
O
O
His345 (334)
Gly114 (114)
Leu115 (115)
Gly640 (629)
Glu637 (626)
Tyr538 (527)
Asn449 (437)
Ser639 (628)
3.0 Å
3.1 Å
2.9 Å
2.9 Å
3.6 Å
3.6 Å
3.6 Å
2.7 Å
3.1 Å
3.7Å
2.7Å
2.8 Å
3.2 Å
4.3 Å
3.2 Å

BA
Fig. 1. Close-up structure of the active site of CcStP and proposed interactions with Glc1P bound at the catalytic subsite. (A) The picture
was generated with
PYMOL v.0.99 using X-ray crystallographic coordinates for CcStP with phosphate bound in the active site (Protein Data
Bank entry 2C4M). His334, PLP and phosphate are shown as stick models. (B) The scheme was drawn using the structure of E. coli malto-
dextrin phosphorylase bound to Glc1P (Protein Data Bank entry 1L5V). Numbering of amino acids is for the E. coli enzyme, and correspond-
ing residues of CcStP are given in parentheses. Hydrogen bonds are indicated as broken lines.
Role of His334 in a-glucan phosphorylase A. Schwarz et al.
5106 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS
lysis or transglucosylation occurs? Mutational analysis
of the His homologous to His334 in CcStP has not
been performed in another a-glucan phosphorylase.
Results
Protein purification and cofactor analysis
Wild-type CcStP and the H334G mutant were pro-
duced in Escherichia coli and purified to apparent
homogeneity (data not shown). Both enzymes were
obtained in similar yields of about 50%, and contained
approximately 0.8 PLPs per subunit of protein. Upon
excitation at 330 nm, the wild-type enzyme and the
H334G mutant exhibited nearly superimposable cofac-
tor fluorescence emission spectra between 470 and
550 nm, with an emission maximum at 520 nm. How-
ever, the intensity of cofactor fluorescence at 520 nm
in the H334G mutant was only approximately 40%
that observed in the wild-type.
Characterization of the H334G mutant
Enzyme activity
The H334G mutant exhibited 0.003% of the wild-type
specific activity for phosphorolysis of maltodextrin

(33 UÆmg
)1
). External imidazole stimulated activity of
the mutant up to 5.5-fold, whereas it weakly inhibited
the wild-type (Fig. 2). Acetate and formate had no
effect on the activity of the H334G mutant. Azide,
2-methylimidazole and 2-ethylimidazole inhibited the
mutant. The wild-type was inhibited weakly (£ 2-fold)
by all of the compounds tested, with the exception of
formate, which caused a five-fold reduction of activity.
Kinetic parameters
Steady-state kinetic parameters for phosphorolysis of
maltopentaose by the H334G mutant were determined
at pH 7.0 under conditions where the concentration of
phosphate was constant and saturating (50 mm). The
k
cat
of 0.033 ± 0.001 s
)1
was 0.05% of the wild-type
value. The K
m
for maltopentaose was 280 ± 20 mm,
reflecting a 75-fold decrease in substrate binding affin-
ity as a result of the mutation. Like the wild-type [18],
the H334G mutant did not hydrolyze maltopentaose
into glucose above a detection limit of about 0.15% of
its phosphorylase activity.
Ligand binding
Dissociation constants (K

d
) for complexes of the
H334G mutant with GL or Glc1P were obtained from
nonlinear fits of a Langmuir binding isotherm to data
obtained by fluorescence titration analysis. The K
d
values were 95 ± 8 lm and 100 ± 10 lm for complexes
with Glc1P and GL, respectively. They were decreased
seven-fold and three-fold in comparison to K
d
values
for corresponding complexes of the wild-type [18]. The
presence of 50 mm phosphate promoted a 30-fold
increase in K
d
(¼ 2.9 ± 0.3 mm) for GL binding to the
H334G mutant. This result is in contrast to the 17-fold
enhancement of GL binding to the wild-type upon the
addition of the same concentration of phosphate.
pH profiles
The pH dependences of logarithmic rates of the
H334G mutant and wild-type are compared in Fig. 3.
Data for the wild-type are taken from Griessler et al.
[19]. The pH profile of the H334G mutant in the phos-
phorolysis direction was a narrow, bell-shaped curve,
strikingly different from that of the wild-type and with
an optimum pH of 6.5. A shift of the pH profile of
about + 0.5–1.0 pH units and an optimum pH similar
to that of the wild-type was observed for the H334G
mutant in the presence of 200 mm imidazole. By con-

trast, the pH rate profile of the wild-type was not
affected by addition of the same concentration of imid-
azole (data not shown).
Phosphorus NMR of pyridoxal 5¢-phosphate
31
P-NMR spectra for solutions of wild-type CcStP and
the H334G mutant that contained a similar concentra-
tion of enzyme-bound PLP ( 100 lm) were recorded
in the pH range 5.6–8.0. Typical spectra acquired at
pH 7.25 are shown in Fig. 4A. The
31
P resonance
imidazole (mM)
0
rel. activity (-fold)
0
2
4
6
100 200
300
400 500
Fig. 2. Analysis of restoration of activity in wild-type CcStP (d) and
the H334G mutant (s) by external imidazole. The results are given
as relative specific activities that were normalized by using the spe-
cific activities of the wild-type (33 UÆmg
)1
) and the H334G mutant
(0.001 UÆmg
)1

) in the absence of imidazole.
A. Schwarz et al. Role of His334 in a-glucan phosphorylase
FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5107
signal of PLP phosphate in the H334G mutant showed
a very low signal-to-noise ratio, necessitating data col-
lection for up to 12 h, during which time a perceptible
denaturation of the enzyme occurred at pH values
below and above 7.25. It was therefore not possible to
obtain an exact pH dependence for the
31
P chemical
shift of PLP phosphate in the H334G mutant. How-
ever, a single
31
P shift at pH 7.25 is provided.
Figure 4B compares pH profiles of chemical
31
P
shifts for PLP phosphate in wild-type CcStP measured
in the absence and presence of 20 mm sodium arsenate.
The two pH profiles were almost superimposable on
each other. We also determined chemical
31
P shifts at
pH 6.68 and 6.93 under conditions in which the pres-
ence of arsenate (20 mm) and GL (1 mm) drives
formation of a ternary enzyme–ligand complex. The
results show that
31
P shifts for PLP phosphate in free

enzyme were remarkably insensitive to the binding of
arsenate alone and in combination with GL.
Analysis of ligand binding by STD NMR
Figure 5 summarizes relative saturation transfer differ-
ence (STD) effects of Glc1P and a-d-xylose 1-phos-
phate (X1P) upon their binding to wild-type and
H334G phosphorylase. Glc1P displayed very similar
patterns of binding to both enzymes. However, the
relative STD effects of the protons in positions 6a
and b were slightly higher when Glc1P was bound to
the wild-type than when it was bound to the H334G
mutant. The relative STD effects of X1P bound to
the two enzymes were also fairly similar, with the
exception of the proton in position 5eq, which showed
a higher effect in the complex with the H334G
mutant. Binding of GL to the wild-type and the
H334G mutant also yielded very similar STD spectra
with, however, quite a low signal-to-noise ratio, very
likely caused by the small dissociation constants for
enzyme–GL complexes. Appreciable STD effects could
be detected only for protons in positions 2 and 4,
which caused overlapping signals in the
1
H-NMR
spectrum (data not shown) [20]. All other protons
showed much lower STD effects, which could not be
quantified. Although longer STD measurements could,
in principle, improve the signal-to-noise ratio, the
duration of the NMR experiment was limited in this
case by the spontaneous hydrolysis of GL to gluconic

acid. During STD NMR measurements of Glc1P
bound to wild-type enzyme, we observed formation of
a novel carbohydrate at the expense of Glc1P. This
compound was analyzed directly from the NMR
sample, and identified as amylose (data not shown).
Details underlying the conversion of Glc1P in the
pH
5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
31
P (p.p.m.)
1
2
3
4
AB
Fig. 4. Characterization of PLP phosphate in
wild-type CcStP and the H334G mutant
using
31
P-NMR. (A) Spectra of wild-type
CcStP and the H334G mutant acquired at
pH 7.25, and with the number of recorded
scans and resulting signal-to-noise ratios
indicated. (B) Chemical
31
P shifts of the PLP
phosphate resonance signal of wild-type
enzyme in the absence of ligand (d), in the
presence of 20 m
M arsenate (s), and in the

presence of 20 m
M arsenate and 1 mM GL
(.);
31
P shift for the H334G mutant (,),
recorded in the absence of arsenate and at
only a single pH of 7.25.
p
H
5.5
log(rel. k
cat
)
1.4
1.6
1.8
2.0
6.0 6.5 7.0 7.5 8.0
Fig. 3. pH profiles of catalytic rates for phosphorolysis of maltodex-
trin catalyzed by wild-type CcStP (.) and the H334G mutant in the
absence (d) and presence (s) of 200 m
M imidazole. The initial
rates were acquired under conditions of apparent saturation with
substrate, and are given as relative values (rel. k
cat
) of the catalytic
rate for the wild-type (50 s
)1
; pH 7.0) and the catalytic rates of the
H334G mutant in the absence (0.0015 s

)1
; pH 6.5) and the pres-
ence (0.0081 s
)1
; pH 7.0) of imidazole. The lines indicate the trend
of the data.
Role of His334 in a-glucan phosphorylase A. Schwarz et al.
5108 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS
absence of an exogenous glucosyl acceptor oligosac-
charide were not pursued further.
Discussion
Disruptive effects of active site mutations traced
by STD NMR
Interpretation of the functional consequences of
H334G and active site mutations of enzymes in general
is subject to the caveat that site-directed replacement
has caused a global change in enzyme–substrate inter-
actions occurring in the wild-type. There is a clear
need for practical methods capable of characterizing
the structural perturbation resulting from site-specific
modification of enzyme or substrate with respect to
direct as well as indirect disruptive effects caused by it.
We would like to suggest the STD NMR technique,
which analyzes, in the dissociated ligand, the magneti-
zation transferred from protons of the protein to pro-
tons of the bound ligand that are in close contact with
the protein. Relative STD effects within a given ligand
therefore provide a characteristic fingerprint of nonpo-
lar ligand interactions within the binding pocket of the
protein [21–27]. Because hydrogen bonds and other

electrostatic interactions are silent in the STD NMR
experiment, the obtained portrait of the binding pat-
tern is partial (Fig. 1B), and isolated interpretations of
STD effects can therefore be hazardous. However, if
STD effects for two minimally modified systems can
be investigated and compared, then the interpretation
is considerably simplified. The side chain of His334
and the –CH
2
OH group of Glc1P are complementary
interacting groups (Fig. 1B), and analysis of changes
in relative STD effects resulting from structural pertur-
bation of enzyme (H334G) and substrate (X1P) was
therefore of particular interest. The results obtained
suggest an overwhelmingly local disruption of binding
interactions caused by removing the two functional
groups individually or together.
Analysis of kinetic consequences in the H334G
mutant and chemical rescue studies
Substitution of His334 with Gly caused a 10
3.5
-fold
decrease in the wild-type k
cat
for phosphorolysis of
maltopentaose. Conversion of the ternary enzyme–sub-
strate complex is believed to be the rate-determining
step of glucosyl transfer to phosphate catalyzed by
a-glucan phosphorylases [1], and k
catP

is the kinetic
measure of it. Because substrate binding to enzyme–
phosphate is supposed to be a rapid equilibrium pro-
cess [1,28], the K
m
for maltopentaose is an effective
dissociation constant that was increased by almost two
orders of magnitude in the H334G mutant in relation
to the wild-type. Comparison of different CcStP
mutants reported here (H334G) and in a recent paper
(H334A, H334Q, H334N [18]) reveals that complete
removal of the His side chain in the H334G mutant
had the largest disruptive effect on both binding and
turnover of maltopentaose. Unlike the H334A mutant,
in which the kinetic consequences of the site-directed
replacement were minimal [18], the H334G mutant had
lost  30 kJÆmol
)1
of the binding energy used in the
wild-type for stabilization of the transition state of the
reaction. (The differential binding energy DDG# was
Fig. 5. Analysis of sugar 1-phosphate bind-
ing to wild-type CcStP and the H334G
mutant using STD NMR. Values are relative
STD effects of Glc1P bound to wild-type
CcStP (Aa) and the H334G mutant (Ab) as
well as X1P bound to wild-type CcStP (Ba)
and the H334G mutant (Bb). Each STD
effect is calculated as a quotient of signal
intensities in the STD spectrum and in the

reference proton spectrum. The effects are
normalized to the respective largest effect
in the sample.
A. Schwarz et al. Role of His334 in a-glucan phosphorylase
FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5109
calculated with the relationship DDG# ¼ RT ln 10
5.2
,
using the ratio of k
catP
⁄ K
m
values of 18 000 m
)1
Æs
)1
and 0.12 m
)1
Æs
)1
for the wild-type and the H334G
mutant, respectively.) We speculated that water might
occupy the position vacated in the H334A mutant
through removal of the imidazole group of the His,
thereby effectively replacing the function of the origi-
nal side chain in catalysis by the mutant [18]. What-
ever mechanism truly accounts for the retention of
phosphorylase activity by the H334A mutant, it is
clearly not available to the H334G mutant. The selec-
tivity of the H334G mutant for glucosyl transfer to

phosphate as compared with water was absolute within
the limits of detection of the experimental methods,
suggesting that, as in the wild-type and the H334A
mutant [18], water was effectively excluded from the
reaction with maltopentaose bound to free enzyme or
enzyme–phosphate.
The notion that substitution of His334 with Gly
destabilizes the transition state of glucosyl transfer but
otherwise does not alter the course of the reaction cat-
alyzed by CcStP is further supported by the results of
linear free energy relationship (LFER) analysis and
chemical rescue studies. Schwarz et al. [18] have shown
that a log–log correlation of catalytic efficiencies of the
wild-type and His334 mutants for phosphorolysis of
starch with the corresponding reciprocal dissociation
constants for GL binding to enzyme–phosphate was
linear, with a good coefficient of determination. Using
a similar type of correlation, which is now based on
k
catP
⁄ K
m
for maltopentaose and includes data for the
H334G mutant, we obtain again a plausible LFER
with a slope of 1.93 ± 0.45 and a coefficient of deter-
mination (r
2
) of 0.862 (supplementary Fig. S1). A shift
in the controlling mechanism of the reaction brought
about by the His fi Gly mutation would be expected

to cause a breakdown of the LFER, in contrast to the
observations made. Whereas external imidazole weakly
enhanced the activity of the H334G mutant, it did not
participate in the reaction as alternative nucleophile,
such that glucose 1-imidazole or the product of its
spontaneous hydrolysis (glucose) would be formed in
kinetic competition with Glc1P. Other small nucleo-
philes, such as azide, were without effect on both
activity and reaction course. By way of comparison,
when the catalytic nucleophile (Asp) of sucrose phos-
phorylase was replaced by Ala, azide could occupy the
position of the original carboxylate group and react
through addition to C-1 of the glucosyl moiety, yield-
ing the inversion product b-glucose 1-azide [9].
We investigated whether the proposed hydrogen
bond between His334 and the C-6 hydroxy group of
the glucosyl residue bound at the catalytic subsite
could become optimized in the transition state. A
hypothetical scenario, inspired by studies of human
purine nucleoside phosphorylase [29,30], is that His334
could be responsible for positioning O-6 in line with
O-5 and the glycosidic oxygen of phosphate (O
P1
)
(Fig. 6). In the direction of polysaccharide synthesis,
compression of the three-oxygen stack such that O-6
moves closer to the ring oxygen would enhance elec-
tron density in the reactive carbon and thus facilitate
glycosidic bond cleavage and formation of the transi-
tion state in an S

N
i-like mechanism of glucosyl trans-
fer. In the direction of phosphorolysis, both O-6 and
the now nucleophilic O
P1
of phosphate might be
pushed towards O-5 and assist electronically in cataly-
sis. As in purine nucleoside phosphorylase [29,30],
protein vibrations that are coupled to the reaction
coordinate could be responsible for promoting the
close approach of the three oxygens.
pH rate dependences for the wild-type and the
H334G mutant examined with kinetics and
31
P-NMR
As for other a-glucan phosphorylases [31–35], the pH
profiles of apparent k
cat
for wild-type CcStP were
ND-1
2.7 Å
2.3 Å
3.0 Å
3.8 Å
O-6
O-5
O
P1
Fig. 6. Suggested role for the hydrogen bond between Nd of
His334 and the 6-OH of the glucosyl residue bound at the catalytic

subsite in the selective stabilization of the transition state. O-6,
the ring oxygen, and the glycosidic oxygen O
P1
lie in a close three-
oxygen stack that is indicated by a dashed line. Increased electron
density near the reactive center provided by squeezing the three
oxygens together could facilitate the catalytic step. The picture was
generated using Protein Data Bank entry 1L5V (maltodextrin phos-
phorylase bound with Glc1P [15]).
Role of His334 in a-glucan phosphorylase A. Schwarz et al.
5110 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS
bell-shaped curves showing a decrease in activity at
low and high pH. Replacement of His334 with Gly
caused a marked change in the pH profile of k
cat
for
the phosphorolysis direction. To explore possible
sources of the different pH dependences, we used
31
P-NMR and compared chemical shifts for the
5¢-phosphate group of PLP in the wild-type and the
H334G mutant. Changes in chemical shift and line
width of the
31
P-NMR signal may serve as reporters
of alterations in the ionization state of the cofactor
phosphate group [36]. They are, however, also expli-
cable by changes in the local environment of PLP
and their effect on conformational strain on the
5¢-phosphate moiety.

The
31
P chemical shift of PLP phosphate in unli-
ganded wild-type CcStP was strongly influenced by pH,
increasing in a sigmoidal dependence from 1.36 p.p.m.
at pH 5.6 to 3.66 p.p.m. at pH 8.0 (Fig. 4B). Slow
deprotonation of the triethanolamine buffer interfered
with measurement of
31
P chemical shifts in the alkaline
region (pH > 7.5), preventing determination of a
complete pH profile for the chemical shift and hence
calculation of the pK
a
value of PLP phosphate by curve
fitting. However, there is good evidence that the pK
a
value for PLP phosphate in free Cc StP is ‡ 6.75
(Fig. 4B), and therefore higher than that seen in
maltodextrin phosphorylase (pK
a
¼ 5.6) [37,38].
To the extent that the shift of the
31
P resonance
signal is a sensitive probe of direct contacts between
the cofactor 5¢-phosphate group and bound ligands
or relevant changes in active site conformation
induced by ligand binding [2,38], the evidence for
CcStP suggests that the local environment of PLP

phosphate remains essentially unaffected upon forma-
tion of enzyme complexes with arsenate alone and in
combination with GL. By contrast, significant field
shifts of the
31
P resonance signal were observed with
E. coli maltodextrin phosphorylase [37], potato phos-
phorylase [39] and muscle glycogen phosphorylase
[40] upon addition of arsenate, probably caused by
electrostatic interactions between the 5¢-phosphate
moiety and arsenate. The pK
a
for PLP phosphate in
E. coli maltodextrin phosphorylase was also shifted
by + 1.1 pH units upon binding of arsenate [37].
Therefore, CcStP appears to differ subtly from mal-
todextrin and glycogen phosphorylase in how it copes
with constraining the cofactor phosphate group into a
configuration that is believed to promote catalysis via
direct interaction with the substrate arsenate (or
phosphate). A tentative explanation is provided by
Fig. 7, which reveals clear differences in the pattern
of hydrogen bonding and the orientation of PLP
phosphate in the active sites of CcStP bound with
phosphate (Fig. 7A) and maltodextrin phosphorylase
bound with phosphate and a nonphosphorolyzable
substrate analog (omitted in Fig. 7B for reasons of
clarity) in Fig. 7B. Gly642 in the E. coli enzyme is
substituted by Ser631 in CcStP. Interactions from the
main chain amide of Gly are replaced by interactions

from both the main chain amide and the side chain
of Ser. Hydrogen bonds between PLP phosphate and
the side chains of nearby Lys residues and bound
phosphate ligand appear to be stronger in maltodex-
trin phosphorylase than in CcStP, arguably account-
ing for the relative elevation of pK
a
of PLP
phosphate in unliganded CcStP and the apparent lack
of perturbation of pK
a
in the CcStP complex with
arsenate.
The pH dependence of the
31
P chemical shift of PLP
phosphate in wild-type CcStP is not, clearly, borne out
in pH rate profiles for the enzymatic reaction. The opti-
mum pH range for glucosyl transfer to and from phos-
phate overlaps with the pH region (pH 6.0–7.0) where
monoanionic and dianionic forms of 5¢-phosphate
should both be present in similar relative amounts. The
loss of wild-type activity in the direction of synthesis at
A B
Fig. 7. Comparison of the sites for PLP
phosphate in CcStP (A) and E. coli malto-
dextrin phosphorylase (B). Pictures were
generated using Protein Data Bank entries
2C4M (CcStP) and 1L5W (maltodextrin
phosphorylase bound with phosphate and a

substrate analog [15]).
A. Schwarz et al. Role of His334 in a-glucan phosphorylase
FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5111
pH 6.5 [19] may be correlated, at least formally, with
the strong field shift of
31
P resonance signal in this pH
range, perhaps reflecting the formation of a PLP dian-
ion. Electrostatic repulsion may now prevent the cofac-
tor 5¢-phosphate and also the dianionic phosphate of
the glucosyl donor substrate from closely approaching
each other [2,41].
Rather than eliminating a single ionization from
pH profiles, substitution of His334 by Gly caused a
complex pattern of changes in the pH rate dependenc-
es of the wild-type. The acidic and basic limbs on the
pH profile of the H334G mutant for the phosphoroly-
sis direction were displaced inward by  0.5 pH units
in comparison with the corresponding pH profile of
the wild-type, and the optimum pH range for the
mutant was also shifted, by about ) 0.75 pH units. In
addition to partly restoring activity in the H334G
mutant, external imidazole caused an upshift by £ 1.0
pH units of the entire pH dependence of phosphoro-
lysis by the mutant, whereas the pH rate profile of
the wild-type was not influenced by the added imidaz-
ole. Although these results suggest that His334 influ-
ences the pH dependence of the activity of CcStP,
they do not delineate a detailed relationship. Appar-
ent ionizations on the pH rate profiles must probably

be assigned to pH-dependent ‘titration’ of more than
just a single residue.
Experimental procedures
Materials
Materials for mutagenesis, protein purification and enzy-
matic assays have been described elsewhere in more detail
[18,42]. Restriction endonucleases were obtained from Fer-
mentas (St Leon-Rot, Germany). Oligonucleotide synthesis
and DNA sequencing was performed at VBC Biotech Ser-
vices GmbH (Vienna, Austria). All other chemicals were of
the highest quality and were provided by Sigma-Aldrich
(Vienna, Austria).
Mutagenesis, protein expression and purification
The point mutation His334 fi Gly was introduced by the
PCR-based overlap extension method [43]. PCR conditions
were as described previously [42], except for an annealing
and elongation time of 1 min and an annealing temperature
of 55 °C. We used the following pairs of flanking and
mutagenic primers, where Eco91I and XagI restriction sites
are underlined and the mismatched codons are indicated
in bold, respectively: XagI-for, 5¢-GGGAACTCTGCG
CCT
GTGGAAGGC-3¢; Eco91I-inv, 5¢-CTCATCCAGATCG
GTTACCCAATC-3¢; H334G-for, 5¢-TACACCAACGGAA
CCGTGCTCAC-3¢; H334G-inv, 5¢-GTGAGCACGGTTC
CGTTGGTGTACGC-3¢.
The plasmid pQE70–CcStP [42] containing the gene for
wild-type CcStP was used as the template. The mutagenized
plasmid was transformed into E. coli JM109 cells. Protein
expression and purification of the H334G mutant were car-

ried out using published protocols [18]. Enzyme activity
was measured with a continuous coupled assay reported
elsewhere [9], and protein was determined by the Bio-Rad
(Vienna, Austria) dye binding assay using BSA as standard.
Steady-state kinetic analysis and biochemical
characterization
Initial rates of phosphorolysis were determined in discontin-
uous assays as described previously [44]. The enzyme (5.5 lm
subunits of the H334G mutant) was incubated at 30 °Cin
300 mm potassium phosphate buffer, and the release of
Glc1P was measured as a function of time of incubation up
to 3 h. Maltodextrin or maltopentaose was used as the sub-
strate, as indicated in Results. The sodium salts of azide, ace-
tate, and formate, as well as imidazole, 2-ethylimidazole, and
2-methylimidazole, were tested in the range 10–250 mm for
possible restoration of activity of the H334G mutant for
phosphorolysis of maltodextrin (23 gÆL
)1
) at pH 7.0. Con-
trol reactions with the wild-type were carried out in all cases.
The H334G mutant was examined for possible hydrolase
activity by incubating the enzyme (6.7 lm)at30°Cin
50 mm triethanolamine buffer (pH 7.0), containing malto-
pentaose (75 mm) and potassium sulfate (20 mm). Note that
sulfate was added in this series of measurements to ensure
stability of the enzymes during the timespan of experiments
carried out in the absence of phosphate [45]. Samples were
taken at certain time points up to 40 h, and the formation of
glucose was measured as described elsewhere [18].
pH dependence studies were performed in the pH range

5.5–8.0. The pH values were adjusted at the temperature of
measurement (30 °C), and controlled before and after the
enzymatic reaction. Ionic strength changes in the pH range
examined were not considered. Catalytic rates of the
H334G mutant were acquired under conditions of apparent
saturation with substrate (300 mm phosphate, 23 gÆL
)1
mal-
todextrin).
Apparent dissociation constants for the binary complex
of the H334G mutant bound with Glc1P or GL, and for
the ternary complex of the H334G mutant bound with
phosphate and GL, were determined by titration analysis in
which quenching of the PLP fluorescence was measured.
Following excitation at 330 nm, an emission spectrum in
the range 350–550 nm was recorded. The full experimental
protocol and details of data processing for the calculation
of dissociation constants are given elsewhere [18]. The PLP
content of isolated H334G mutant was measured with a
quantitative spectrophotometric test [46].
Role of His334 in a-glucan phosphorylase A. Schwarz et al.
5112 FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS
NMR spectroscopy
All NMR measurements were recorded in
2
H
2
O
(99.9%
2

H) at 30 °C on a Bruker (Rheinstetten, Germany)
DRX 600 AVANCE spectrometer using topspin 1.3 soft-
ware (Bruker). Proton, carbon and phosphorus spectra
were measured at 600.13 MHz, 150.90 MHz, and
242.94 MHz, respectively. The one-dimensional spectra
were recorded with 32 768 data points. Zero filling to
65 536 data points, appropriate exponential multiplication
and Fourier transformation led to spectra with ranges of
 5400 Hz (
1
H),  33 000 Hz (
13
C), and  24 000 Hz (
31
P).
pH-dependent
31
P chemical shifts were determined employ-
ing a slight modification of a reported procedure [37].
Samples were prepared by adding 150 lL of 400–450 lm
enzyme solution in 50 mm triethanolamine buffer, contain-
ing 20 mm potassium sulfate, to 450 l L of a solution con-
taining 50 mm triethanolamine buffer, 50 mm acetate and
20 mm potassium sulfate in
2
H
2
O. At the concentration
used, sulfate does not inhibit the enzyme activity through
competition with phosphate, suggesting that occupancy of

the active site by sulfate is negligible.
31
P spectra were
measured with proton decoupling and 2048 scans and a
preacquisition delay of 1.0 s, resulting in spectra with a sig-
nal-to-noise ratio of about 10 : 1 to 20 : 1 after about 1 h.
Two-dimensional spectra were obtained from 256 experi-
ments, each with 2048 data points and an appropriate num-
ber of scans. Zero filling and Fourier transformation led to
spectra with ranges of  5400 Hz and  30 000 Hz for
proton and carbon, respectively. Chemical shifts have
been referenced to external acetone (d
H
2.225 p.p.m.;
d
C
31.45 p.p.m.) and 85% aqueous phosphoric acid
(d
P
0.00 p.p.m.).
STD NMR spectra were measured employing a reported
procedure [27]. Samples were prepared in 5 mm potassium
phosphate buffer (pH 7.0), containing  10 lm enzyme as
well as 5 mm ligand. Before addition to the NMR tube, the
enzyme storage solution was gel-filtered using NAP 5 col-
umns (GE Healthcare, Vienna, Austria) equilibrated with
5mm potassium phosphate buffer in
2
H
2

O (pH 6.65). Five
hundred and twelve scans were collected, each with 50
Gaussian-shaped pulses (50 ms and 1 ms delay) and a
30 ms spin lock pulse, resulting in spectra of  4200 Hz
spectral width. On and off resonance irradiations were
made at d
H
) 2.00 p.p.m. and d
H
41.66 p.p.m., respectively,
subtraction was performed via phase cycling, and no water
suppression was applied. Reference proton spectra were
recorded with 256 scans directly before and after the STD
measurements.
Acknowledgements
Financial support from the Austrian Science Fund
(FWF P15208-B09, P18038-B09 and P15118) is grate-
fully acknowledged.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Log–log correlation between the reciprocal
dissociation constants of GL binding to enzyme–phos-

phate complexes of wild-type CcStP and His334
mutants and k
cat
⁄ K
m
values for phosphorolysis of
maltopentaose (A) and starch (B).
This material is available as part of the online article
from
Please note: Blackwell Publishing 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.
A. Schwarz et al. Role of His334 in a-glucan phosphorylase
FEBS Journal 274 (2007) 5105–5115 ª 2007 The Authors Journal compilation ª 2007 FEBS 5115