Tài liệu Báo cáo khoa học: Relationships between structure, function and stability for pyridoxal 5¢-phosphate-dependent starch phosphorylase from Corynebacterium callunaeas revealed by reversible cofactor dissociation studies doc

Relationships between structure, function and stability for pyridoxal
5¢-phosphate-dependent starch phosphorylase from
Corynebacterium
callunae
as revealed by reversible cofactor dissociation studies
Richard Griessler, Barbara Psik, Alexandra Schwarz and Bernd Nidetzky
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
Using 0.4
M
imidazole citrate buffer (pH 7.5) containing
0.1 m
ML
-cysteine, homodimeric starch phosphorylase from
Corynebacterium calluane (CcStP) was dissociated into
native-like folded subunits concomitant with release of
pyridoxal 5¢-phosphate and l oss o f activity. The inactivation
rate of CcStP under resolution conditions at 30 °Cwas,
respectively, four- and threefold reduced in two mutants,
Arg234fiAla and A rg242fiAla, previously shown to cause
thermostabilization of CcStP [Griessler, R., S chwarz, A.,
Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem. 270, 2126–
2136]. The proportion of original enzyme activity restored
upon the reconstitution of wild-type a nd mutant apo-phos-
phorylases with pyridoxal 5¢-phosphate was increased up to
4.5-fold by added phosphate. The effect on recovery of
activity displayed a saturatable dependence on the phos-
phate concentration and results from interactions with the
oxyanion that are specific to the quarternary state.
Arg234fiAla and Arg242fiAla mutants showed, respect-
ively, eight- and > 20-fold decreased apparent affinities for
phosphate (K

app
), compared to the wild-type (K
app
 6m
M
).
When reconstituted next to ea ch other in solution, apo-
protomers o f Cc StP and Escherichia coli maltodextrin
phosphorylase did not detectably associate t o hybrid d imers,
indicating that structural complementarity among the dif-
ferent subunits was lacking. Pyridoxal-reconstituted CcStP
was i nactive but  60% and 5% of w ild-type a ctivity could
be rescued a t p H 7 .5 by phosphate (3 m
M
) and phosphite
(5 m
M
), respectively. pH effe cts on catalytic rates were dif-
ferent for the native enzyme and pyridoxal-phosphorylase
bound to phosphate and could reflect the differences in
pK
a
values for the cofactor 5¢-phosphate and the exogenous
oxyanion.
Keywords: apo-phosphorylase; a-glucan; glycogen; malto-
dextrin; pyridoxal 5¢-phosphate.
Structure–function relationship studies of a-glucan phos-
phorylases (GP) have a rich history in biochemical litera-
ture. It i s well established that pyridoxal 5¢-phosphate (PLP)
is the essential cofactor in all known GPs [1]. PLP is bound

via a Schiff b ase between it s aldehyde g roup and a
conserved l ysine s ide c hain in the active s ite [ 1,2]. T he
5¢-phosphate group is a main catalytic component of PLP
and is required for GP activity [2]. The functional oligomeric
state of GP is dimeric [3–5]. It has been shown t hat
dissociation of the subunits under localized denaturing
conditions exposes PLP to solvent. PLP is released from the
enzyme and the activity is lost [6–8]. Apo-phosphorylase can
be reconstituted, either with PLP or a range of structural
analogues thereof [2,9,10]. Whereas restoration of enzyme
activity upon the apofiholo conversion is determined by
cofactor structure, the process of dime rization is relatively
indiscriminate in respect to structural modifications of PLP.
Induction of structural complementarity of the in teracting
subunits such that they are a ble to recognize each other and
associate to d imers i s c orrelated with enzyme–cofactor bond
formation [5,9]. In a thorough i nvestigation, Helmreich and
colleagues prepared a series of hybrid phosphorylases in
which one subunit contained P LP while the other was
bound to an inactive cofactor analogue [5]. They concluded
that intersubunit contacts were als o needed to elicit activity
in a potentially active holo-monomer.
With very few exceptions [11,12], the results just sum-
marized were obtained with a single enzyme, GP f rom
rabbit muscle (RmGP). The a ctivity o f R mGP is under the
control of allosteric and covalent regulatory mechanisms
which are different or completely lacking in a large group of
GPs from plants and microorganisms. We therefore asked
the question, what novel information might be gained by
applying the same type of reconstitution experiments

described for RmGP to another p hosphorylase from a
different source with different regulatory properties? While
active-site residues are almost invariant in members of the
GP family, the dimer interfaces have been quite variable
during the evolution in respect to the specific interproto-
meric contacts, as revealed by comparative 3D structural
Correspondence to B. Nidetzky, Institute of Biotechnology and Bio-
chemical Engineering, Graz University of Technology, Petersgasse 12/
I, A-8010 Graz, Austria. Fax: +43 316 873 8434,
Tel.: +43 316 873 8400, E-mail:
Abbreviations: GP, glycogen phosphorylase; Ec MalP, Escherichia coli
maltodextrin phosphorylase; CcStP, Corynebacterium callunae starch
phosphorylase; PLP, pyridoxal 5¢-phosphate; PL, pyridoxal;
RmGP, rabbit muscle GP.
Enzyme: a-glucan phosphorylase or a-1,4-
D
-glucan:orthophosphate-
a-
D
-glucosyltransferase (EC 2 .4.1.1).
(Received 2 5 March 2004, rev ised 21 June 2004,
accepted 22 June 2004)
Eur. J. Biochem. 271, 3319–3329 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04265.x
[13] and structure-based sequence analyses [14,15]. The
overall c ontact p attern at the subunit i nterfaces of different
regulated an d nonregulated GPs is h owever, w ell p reserved
[13]. Thus one would like to know what directs subunit
interactions towards t he induction of full enzymatic activity
and optimum stability in a dimer of phosphorylase. This is a
significant and central problem to the study of catalysis by

GPs and oligomeric enzymes in general where the individual
subunits seem to possess all of t he requisite chemic al
functions but are in a catalytically inactive and unstable
conformation. The detailed e xamination of the steps
involved in subunit d issociation and reassociation will
contribute to a better understanding of the dimerization
process per se and the role of interprotomeric contacts to
generate a functional enzyme. The utilization of a phos-
phorylase devoid of the complex regulatory mechanisms
seen in RmGP allows the analysis to b e strictly focused on
catalytic activity and stability.
We chose starch phosphorylase from Corynebacterium
callunae (CcStP), which has been characterized biochemi-
cally and structurally [ 15,16], for particular reason. The
intersubunit contacts stabilizing t he functional CcStP dimer
are strengthened by > 100-fold when oxyanions such as
phosphate bind to this enzyme [17]. Enzyme–oxyanion
interactions occur a t a protein site different from the active
site, and thermostabilization is the result of a protein
conformational change induced by the binding event.
Residues involved in t he str uctural rearrangement a re
located within t he predicted d imer contact r egion of Cc StP
[15]. Reversible subunit d issociation experiments should
thus be useful to explore structural requirements for the
phosphate effect on CcStP stability.
We rep ort here the preparation of apo-CcStP and the
characterization thereof in respect to structural properties
and kinetic stability. The process of reconstitution with PLP
has been analyzed using CcStP and four site-specific mutants
in which amino acid replacements within the dimer contact

region have led to a ltered oxyanion-dependent kinetic
stabilities [15,18]. The relative timing of steps involved in
dimer formation and a ppearance of thermostabilization by
phosphate has been examined. The role of the cofactor
5¢-phosphate group in the induction of stability and stabil-
ization of the CcStP dimer has been studied. Subunit
complementation e xperiments are r eported w hich were
designed to detect formation of possible hybrid dimers of
CcStP and maltodextrin p hosphorylase from Escherichia c oli
(EcMalP). Finally, w e s how results from kinetic studies of
CcStP reconstituted with p yridoxal (PL),a cofactor analogue
in which the original 5¢-O-PO
3
2–
group is replaced by 5¢-O-H.
Materials and methods
Enzymes, substrates and other materials
Recombinant CcStP a nd site-directed mutants thereof were
produced as described elsewhere [15,18]. Natural CcStP w as
purified by a reported p rocedure [16]. If not stated
otherwise, recombinant CcStP was used. EcMalP was
prepared accordin g to Eis et al.[19].Analyticalenzymes
and enzyme substrates were specified in previous papers
[15–18]. Al l other chemicals were of reagent grade and
obtained from Sigma and F luka.
Preparation of apo-
Cc
StP and apo-
Ec
MalP

Screening for buffer conditions in which apo-CcStP could
be prepared, led to selection of 0.4
M
imidazole citrate and
0.1
M
cysteine hydrochloride, in short, the resolution buffer.
Various pH values between 5.0 and 8.0 were tested, and a
pH of 7.0 was chosen (see below). Prior to the resolution,
CcStP and site-directed mutants thereof were doubly gel
filtered using NAP 5 or NAP 10 columns (Amersham
Biosciences) to remove phosphate from storage stock
solutions to an end concentration below 0.1 m
M
.The
enzymes were incubated in the resolution buffer at 30 °C
using protein concentrations in the range 0.5–2.0 m gÆmL
)1
until the residual activity w as between 1.5 and 2 .5% o f the
original leve l. The r esolution buffer w as th en replaced by a
50 m
M
triethanolamine buffer, pH 7.0, using gel filtration
with a NAP 5 column. Separate control experiments for
wild-type CcStP showed that the fourfold variation in
protein concentration in our experiments was not an
important factor of the rate of resolution.
Apo-EcMalP was prepared using a protocol developed
by Palm and c oworkers (D. P alm, Theodor-Bover i-Institut
fu

¨
r Biowissenschaften, Univer sita
¨
tWu
¨
rzburg, Germany;
personal communication). T he enzyme was diluted to
2mgÆmL
)1
in 50 m
M
Mes buffer, pH 7.0, containing
25 m
M
KCl and 2 m
M
dithiothreitol. An equal volume o f
1
M
cysteine hydrochloride dissolved in the same buffer w as
added to g ive a final concentration of 0.5
M
.Resolutionwas
obtained by adjusting the p H with HCl to a v alue of 5.05 at
4 °C. The enzyme was incubated under these conditions
until the residual activity was about 1.5% of the original
level. Apo-EcMalP was precipitated by ammonium sul-
phate at 65% saturation, and the pellet w as resuspended in
50 m
M

potassium phosphate buffer, pH 7.0.
The time course of apo-phosphorylase formation was
monitored by using a number of methods [17]: enzyme
activity measurements using s amples taken from the incu-
bation mixture; column sizing experiments t o determine the
subunit association state of the protein; CD spectroscopic
measurements; determination of protein-bound and disso-
ciated PLP. This latter measurement was performed after
ultrafiltration of the sample using 30 kDa cut off micro-
concentrator tubes. The PLP content of the
protein-containing retentate was measured using both
semiquantitative fluorometric analysis and a quantitative
spectrophotometric test [17]. The fi ltrate, w hich was devoid
of protein, was the subject o f quantitative analysis for PLP
content.
Apo-phosphorylases were always prepared for immediate
further use and not stored for longer than about 2 h at 4 °C.
Appropriate control measurements showed that the inacti-
vation of apo-enzymes was not significant under these
conditions.
Reconstitution of apo-phosphorylases
Apo-phosphorylase of Cc StP (about 0.1–0.4 mgÆmL
)1
)was
brought to 50 m
M
triethanolamine buffer, pH 7.0, contain-
ing a concentration o f potassium phosphate between < 0.05
and 80 m
M

. PLP at a concentration of between 0.0 and
100 l
M
was added to reconstitute the holo-enzyme. The
reaction was carried out at 30 °C and typically, the time
3320 R. Griessler et al.(Eur. J. Biochem. 271) Ó FEBS 2004
course of recovery of enzyme activity was monitored up to
180 m in. When addition of fresh PLP did not further
enhance the regain of activity, reconstitution was c onsidered
to be exhaustive. Reconstituted CcStP was characterized in
respect to its structural properties using CD spectroscopy,
cofactor fluorescence and analytical gel filtration using
Superose 12 HR 10/30 (see below). Kinetic parameters of
the direction of a-glucan phosphorolysis and synthesis were
determined as described below. Reconstitution of apo-
EcMalP was performed at 30 °Cin50m
M
potassium
phosphate buffer, pH 7.0, and incubation was carried on
4 h after a ddition of 100 l
M
PLP.
Using th e co nditions described above, a reconstitution
experiment was carried out in which apo-CcStP
(0.35 mgÆmL
)1
of the natural enzyme) and apo-EcMalP
(1.35 mgÆmL
)1
) were incubated with 100 l

M
PLP next to
each other in solution. Therefore, heterodimerization would
have been possible, and the aim was to either detect it or rule
out its occurrence under the conditions used. The protein
solution was loaded on to a 5 mL Econo-Pac column of
ceramic hydroxylapatite type II (Bio-Rad) equilibrated with
50 m
M
potassium phosphate buffer, pH 6.8. Elution was
carried out at room temperature with a step gradient of 1
M
potassium phosphate buffer, pH 6.8, at a flow rate of
 40 cm Æh
)1
. Fractions containing protein were collected,
concentrated using ultrafiltration microconcentrator tubes,
and gel filtered using NAP 10 columns. C haracterization of
the fractions was carried out in respect to: the N-terminal
sequence determined by automated Edman degradation;
stability a t 5 0 °Cwhen0.3
M
potassium phosphate (pH 7.0)
was present; and kinetic parameters for phosphorolysis of
maltohexaose (Sigma) at 30 °C.
Enzyme kinetic measurements
Phosphorylase activity was measured in the direction of
a-glucan phosphorolysis using a continuous, phosphoglu-
comutase and NAD
+

-dependent glucose 6-phosphate
dehydrogenase-coupled spectrophotometric assay, des-
cribed in more detail elsewhere [16]. If not mentioned
otherwise, maltodextrin 19.4 (Agrana, Gmu
¨
nd, Austria) was
the a-glucan s ubstrate. Initial rates of a-glucan phosphoro-
lysis and synthesis were recorded with discontinuous assays,
as reported previously [16]. Linear plots of product
concentration vs. time were converted into rates. Kinetic
parameters were obtained from nonlin ear fi ts of initial r ate
data to Eqn (1) using the
SIGMAPLOT
program (SPSS Inc.,
Chicago, IL, USA),
v ¼ k
cat
½E½S=ðK
m
þ½SÞ ð1Þ
where v is t he i nitial rate, k
cat
is the turnover number, [E] is
the molar c oncentration of enzyme active sites (based on the
stoichiometry of PLP and enzyme subunit), K
m
is an
apparent Michaelis constant, and [S] is the substrate
concentration. When inhibition at high [S] was observed,
Eqn ( 2) was used:

v ¼ k
cat
½E½S=ðK
m
þ½Sþ½S
2
=K
iS
Þð2Þ
where K
iS
is the substrate inhibition constant.
pH effects of enzyme-catalyzed initial rates were recorded
at 3 0 °Cin0.1
M
sodium acetate buffer in the pH range 5.0–
8.0. If not indicated otherwise, it was proved that enzyme
inactivation d uring the time of the discontinuous assay
( 15 min) was n ot a source of an observable pH depend-
ence of activity. pH profiles were fitted t o Eqn (3),
log rate ¼ log½C=ð1 þ K
a
=½H
þ
Þ ð3Þ
where C is the pH-independent value of the rate, K
a
is a
macroscopic acid dissociation constant, and [H
+

]isthe
proton concentration. Equation (3) i mplies a pH profile
that is level below pK
a
and decreases a bove p K
a
with a slope
of )1.
Stability of apo-phosphorylase
Apo-phosphorylase ( 0.2 mgÆmL
)1
) was incubated in
0.1
M
sodium acetate buffer, pH 6.9, at 22 °C . At certain
times between 0.2 and 20 h, samples were taken from the
reaction mixture, PLP (40 l
M
) and potassium phosphate
(50 m
M
) were added, and reconstitution was allowed to
proceed for up t o 4 h b efore recovered enzyme activity was
measured. The activity of the reconstituted phosphorylase
at zero i ncubation time served as the control. A number of
compounds were tested in respect to a potential stabilization
of apo-phosphorylase, and they were added in the c oncen-
trations shown under Results. Pyridoxin 5¢-phosphate was
prepared by reduction of PLP w ith N aBH
4

. Control
experiments were carried out in which pyridoxin 5¢-phos-
phate (2 m
M
)wasincubatedat30°C with apo-phosphory-
lase and regain of activity was recorded over time. T he total
lack of recovery of activity proved that the reduction of PLP
was complete.
Structural characterization
CD spectroscopic measurements were c arried out with a
Jasco J-600 s pectropolarimeter using quartz cuvettes of
0.1 c m pathlength. Spectra of protein samples
( 0.1 mgÆmL
)1
) were recorded at 23 ± 1 °C in the range
200–240 nm. I f not mentioned otherwise, a 50 m
M
potas-
sium phosphate buffer, pH 7.0, was used. Column sizing
experiments were carried out with Superose 12 HR 10/30
(22 mL bed volume) using a 50 m
M
potassium phosphate
buffer, pH 7.0, containing 200 m
M
NaCl and 0.1% (w/v)
NaN
3
. Approximately 200 lg of protein dissolved in 0.5–
1.0 mL of buffer were loaded on to the column, and

elution of protein was detected at 280 n m using an
A
¨
ktaexplorer system (Amersham Biosciences). F luores-
cence measurements were performed with a Hitachi
F-2000 spectrofluorometer using Hellma QS 101 cuvettes.
The excitation wavelength was set to 330 nm, and
emission spectra were recorded in the range 360–600 nm.
Typically, a protein c oncentration of 0 .4 mgÆmL
)1
dissolved in triethanolamine buffer, pH 7.0, was used.
Results
Preparation and characterization of apo-
Cc
StP
Apo-CcStP was obtained at a practically useful rate by
incubating Cc StP in c oncentrations of between 0.5 a nd
2.0 mgÆmL
)1
in 0.4
M
imidazole citrate buffer, pH ¼ 6.8,
Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur. J. Biochem. 271) 3321
containing 0.1
ML
-cysteine h ydrochloride at 30 °C. Loss of
enzyme activity served as the reporter of formation of the
apo-enzyme under these conditions. S emi-logarithmic plots
of the fraction of remaining active Cc StP against time were
linear, suggesting that inactivation can be approximated by

a p seu do first-order m odel. The h alf-life of t he holo-
phosphorylase was  60 min at pH 7.0. The inactivation
rate was pH-dependent and decreased at pH values b elow
6.5. No significant loss of activity was observed at p H 5.0–
5.5 over 1.5 h. When 50 m
M
potassium phosphate or
potassium sulphate was present in the buffer, pH 7.0,
formation of apo-phosphorylase was not detected over a
24 h long incubation time, indicating a half-life of 100 h or
greater. Therefore, stabilization of the native dimer struc-
ture by the oxyanions must be > 1 00-fold (¼ 100/1), in
good agreement with previous results o n the thermostabi-
lization of CcStP [15,17,18].
Column sizing experiments revealed that the apo-phos-
phorylase is a monomer. It does not contain bound PLP
within limits of detection of the denaturing spectrophoto-
metric assay (± 2%). It completely lacks th e characteristic
fluorescence emission o f the cofactor in native CcStP which
occurs in the wavelength r ange 480 –560 nm (see l ater).
Typically, apo-phosphorylases of CcStP and mutants
thereof contained equal to 2% of the original enzyme
activity which can be detected before and after the gel
filtration to replace the resolution buffer.
Figure 1 shows the time course of inactivation of apo-
CcStP at 22 °C in the absence and presence of potential
stabilizers. The half-life o f a po-phosphorylase was approxi-
mately 15 h, and w e observed only small effects o n stability
of added phosphate, sulphate, and the cofactor derivative
pyridoxin 5¢-phosphate. By contrast, UDP-a-

D
-glucose
conferred substantial extra stability to CcStP. ADP-a-
D
-
glucose stabilized apo-CcStP to about the same extent as
UDP-a-
D
-glucose (not shown). Gel filtration analysis o f
apo-CcStP was carried out under c onditions in which UDP-
a-
D
-glucose (1 m
M
) was added to the elution buffer. The
apo-enzyme eluted as a single protein peak and with a
retention time expected f or a monomer of  90–100 kDa.
Therefore, the stabilizing effect of UDP-a-
D
-glucose is
clearly not due to formation of an a po-oligomer induced by
the b inding of the nucleotide sugar. The presence of
maltopentaose (5 m
M
) resulted in a moderate 1.5-fold
increase in the half-life of apo-CcStP.
Effects of mutations in the dimer contact region
on the rate of apo-enzyme formation
The p seudo fi rst-order rate constants of inactivation in
resolution bu ffer at pH 7.0 were determined for CcStP and

five m utant s thereof, using straight-line fits of the d at a
plotted as logarithmic fraction of residual activity vs. time.
The r esults are s ummarized in Table 1. C omparison of rate
constants shows that the effect of the mutation may be
stabilizing (R234A, R242A), neutral (S238A, S224A), or
destabilizing (R226A), compared to the wild-type. Except
for R226A and R242A mutants (Table 1), all enzymes were
stable for 2 h in the presence of 5 m
M
potassium phosphate
and potassium sulphate.
Reconstitutions with PLP of apo-
Cc
StP and mutants
thereof, and characterization of the wild-type
holo-enzyme
Incubation of apo-CcStP ( 0.2 mgÆmL
)1
;2.2l
M
enzyme
subunits) a t 30 °Cin50 m
M
triethanolamine buffer, pH 7.0,
containing 50 m
M
potassium phosphate led to a gradual
regain of enzyme activity in a PLP concentration-dependent
manner. Nine levels of PLP between 2 and 100 l
M

were
tested, a nd the activity r ecovered afte r a 90 min incubation
(which was shown to be exhaustive) displayed a saturatable
dependence on [PLP], with h alf-saturation being attained at
K
PLP
¼ 19 ± 2 l
M
. The recovery of activity when no PLP
was added was not significant within the experimental error
(± 1–2%). To prevent nonspecific reactions of the aldehyde
group of PLP with protein lysines other than Lys634, a
concentration of 2· K
PLP
was chosen for standard recon-
stitution.
Column sizing experiments revealed that reconstituted
CcStP e xisted exclusively as a dimer. CD an d cofactor
fluorescence emission spectra of native and reconstituted
Fig. 1. Stability and stabilization of apo- CcStP. The apo-enzyme
( 0.2 mgÆmL
)1
)wasincubatedat22°Cin0.1
M
sodium acetate
buffer, pH 6.9. Incubations were carried out without additive (d);
5m
M
potassium ph osphate (s); 5 m
M

sodium sulphate ( .); 2 m
M
pyridoxin 5¢-phosphate (,); and 5 m
M
UDP-a-
D
-glucose (j). Activity
in samples taken at the t imes indica ted was measured after reconsti-
tution with 40 l
M
PLP and 50 m
M
potassium phosphate as described
under Materials and metho ds.
Table 1. Half-lives (t
1/2
)ofCcStP and mutants thereof in the resolution
buffer at 30 °C and pH 7.0. Stable,noinactivationwith2hofin-
cubation.
Protein
t
1/2
(min)
No oxyanion 5 m
M
Sulphate 5 m
M
Phosphate
Wild-type 57 ± 4 Stable Stable
S224A 40 ± 4 Stable Stable

R226A 11 ± 0.5 100 ± 10.5 30 ± 4
R234A 260 ± 25 Stable Stable
S238A 36 ± 4 Stable Stable
R242A 190 ± 10 Stable 300 ± 20
3322 R. Griessler et al.(Eur. J. Biochem. 271) Ó FEBS 2004
CcStP and apo-Cc StP are sh own in Fig. 2. T he CD spectr a
of the three proteins are very similar overall, indicating
similarity in respect to the relative composition of secondary
structural elements. However, the characteristic minima in
ellipticity at 208 nm and 222 nm have greater intensities in
the native enzyme, suggesting partial loss of a-he lical
structure in apo-CcStP and reconstituted holo-CcStP. Data
presented in F ig. 2B proves that PLP is incorporated into
apo-CcStP during reconstitution. However, the intensity of
cofactor fluorescence in the reconstituted enzyme is
approximately 65% that observed in CcStP, and this
difference agrees with differences in specific activities of
native and reconstituted phosphorylase. Likewise, cofactor
stoichiometry is decreased from a value of  1 in the wild-
type to  0.6 in the reconstituted enzyme. Apparent
Michaelis constants of reconstituted CcStP were determined
in 50 m
M
triethanolamine buffer, pH 7.0, for phosphate
(4.0 ± 0.3 m
M
); and maltodextrin (3.9 ± 0.4 m
M
)inthe
direction of phosphorolysis; a-

D
-glucose 1-phosphate
(1.0 ± 0.1 m
M
); and maltodextrin (33 ± 5 m
M
)inthe
direction of synthesis. After correction of t urnover numbers
for the fraction of active enzyme in holo-phosphorylase,
native and reconstituted CcStP are not distinguishable in
regard to their kinetic properties.
The time courses of recovery of enzyme activity upon
reconstitution of wild-type and mutant apo-phosphorylases
with 40 l
M
PLP were biphasic. During the initial burst
phase which was complete within 5 min, t here appeared up
to 80% of the total enzyme activity recoverable under the
conditions. In the second phase, enzyme a ctivity i ncreased
slowly to its final level and eventually decreased again.
Figure 3 shows t ypical profiles of regain of a ctivity vs. time
of reconstitution, obtained with the R226A mutant in the
absence a nd presence of potassium phosphate. In all cases
except for the R242A mutant, the yield of enzyme activity
(compared to the original level before resolution and
expressed as a percentage thereof) was increased by added
phosphate (Tab le 2). The effect of phosphate was composed
of two components: first, a shift of apparent equilibrium for
the reconstitution reaction towards t he active enzyme and
second, a stabilization of t he reconstituted h olo-enzyme

against inactivation (which was shown to be irreversible).
We compared recovery of activity of the wild-type under
conditions in which phosphate (50 m
M
) was present from
the b eginning of the r econstitution or was added at the e nd
of the burst phase (5 min). The yield was the same in both
experiments within t he experimental error. The r ecovery of
activity showed a saturatable dependence on t he phosphate
concentration. Half-saturation constants for phosphate
(K
dPi
) were obtained from nonlinear fits of values of final
Fig. 2. Comparison of spectral properties of native CcStP, a po-CcStP,
and reconstituted e nzyme using CD (A) and fluorescence (B). Spe ctra
were recorded using approximately the same protein concentration
(0.1 mgÆmL
)1
± 5 %) in each case. ( A ) Sp ectra of t he native Cc StP
(j), the apo-CcStP (d), and the enzyme after exhaustive reconstitution
inthepresenceof100l
M
PLP (,). (B) Th e fluorescence em ission
spectra are show n for native enzyme (––), apo-CcStP ( ÆÆÆ Æ), and
reconstituted e nzyme (- - -). The excitation wavelength was constant at
330 n m. In (A) and (B), the reconstituted en zyme showed  65% of
the original activity. A 50 m
M
potassium phosphate buffer, pH 7.0,
was used.

Fig. 3. Reconstitution of apo-enzyme of R226A mutant. The assays
contained 0.22 m gÆmL
)1
protein and used 40 l
M
PLP. Other condi-
tions are reported un der Materials an d methods. T he symbo ls show
the different concentratio ns of phosphate in m
M
,asindicated.
Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur. J. Biochem. 271) 3323
recovered activity to Eqn (4) and are summarized in
Table 2 . T hey reveal marked d ecreases i n t he apparent
affinities of the R234A and R242A m utants for phosphate,
compared to wild-type.
DEA ¼ DEA
max
½P
i
=ðK
dPi
þ½P
i
Þ ð4Þ
where DEA is the difference in recovered enzyme a ctivity in
the presence and absence o f phosphate, and DEA
max
is the
maximum value for DEA when phosphate is saturating.
Reconstitutions of apo-

Cc
StP and apo-
Ec
MalP next
to each other in solution
Figure 4 shows fractionation by hydroxylapatite chroma-
tography of a protein mixture obtained by reconstitutions of
apo-CcStP and apo-EcMalP under conditions that might
enable subunit complementation to form a hybrid phos-
phorylase. Through elution with an increasing phosphate
concentration, two major fractions A and C we re isolated
which together accounted for more t han 95% of the total
protein loaded on to the column. It is noteworthy that
fractions A and C eluted exactly as expected for native
CcStP and EcMalP, respectively. Likewise, CcStP and
EcMalP prepared by reconstitution of the corresponding
apo-phosphorylases independent of one another displayed
 70% of their o riginal phosphorylase activities and e luted
exactly a s the native enzymes did (data not shown). Figure 4
shows that a minor fraction B was also obtained. Like
fractions A and C, i t c ontained phosphorylase activity.
Control e xperiments showed that under the conditions used,
the fractionation of reconstituted EcMalP may yield a small
fraction B depending on the applied amount of protein.
Protein fractions A–C were characterized functionally and
structurally, as summarized in Table 3.
Production and characterization of PL-reconstituted
Cc
StP
PL could r eplace PLP i n the reconstitution of apo-CcStP.

The formation of PL-phosphorylase after an exhausti ve
incubation time of  4 h showed a saturatable dep endence
on PL concentration, the optimum level of PL being
approximately 250 l
M
. Addition of PLP (40 l
M
)aftera4h
incubation of apo-CcStP (0.3 mgÆmL
)1
) in the presence of
PL (250 l
M
) did not restore further enzyme activity,
suggesting that reconstitution with PL was complete.
PL-phosphorylase was as stable as the native e nzyme or
PLP-reconstituted CcStP at 60 °Cin300m
M
potassium
phosphate buffer, pH 7.0. Therefore, the cofactor phos-
phate group is not a component of oxyanion-dependent
thermostabilization of CcStP.
When assayed in the direction of a-glucan synthesis at
30 °C (using conditions described in Fig. 5), PL-phosphory-
Table 2. Effect of phosphate on recovered enzyme a ctivity during
reconstitution of apo-enzymes of wild-type CcStP and mutants t hereof
with 40 l
M
PLP. A50m
M

triethanolamine buffer, p H 7.0, w as used.
K
dPi
is the half-saturation c onstant for phosphate. The protein con-
centrations used varied in t he range 1–4 l
M
of ap o-enzyme (9 0 k Da)
and were ‡ 10· th e concen tration o f co factor. Con trol exp eriments
carried out with the wild-type showed that the y ield o f recon stituted
enzyme activity did not change as result of this variation in protein
concentration . The values in parentheses show the yield of recovere d
enzyme activity when no phosphate w as present. ND , not determined,
because no significant dependence of recovered enzyme activity on
[phosphate] was seen in the ran ge 0–80 m
M
.
Protein K
dPi
(m
M
)
Recovered enzyme
activity (%)
Wild-type 6.07 ± 1.29 66 (37)
S224A 2.89 ± 0.51 59 (35)
R226A 10.4 ± 3.8 85 (48)
R234A 47.1 ± 4.5 68 (15)
R242A ND 56 (48)
Fig. 4. Fractionation by hydroxylapatite chromatography of a protein
mixture obtained by r econstitution of apo-CcStP and apo-EcMalP. The

protein e lution profile, recorded by absorbance a t 280 nm, i s shown.
The dashed lin e indic ates the e lution gradie nt used . See Mate rials and
methods for details.
Table 3. Characterization of protein species obtained t hrough c hroma-
tographic fractionation of a mixture of apo-CcStP and apo-EcMalP
reconstituted with 100 l
M
PLP next to each other in solution. Figure 4
gives details of the fractionation. Fractions are labeled according to
Fig. 4. K
mG6
and K
iG6
were obtained from nonlinear fi ts to Eqn (2) of
the initial rate data recorded at a c onstant s aturating c oncentration o f
50 m
M
P
i
. K
mG6
and K
iG6
are the apparent Michaelis constant and the
substrate inhibition constant for m altohexaose, respectively. Half-life
(t
1/2
) incubations were carried out at 50 °Cin300m
M
potassium

phosphate buffer, pH 7.0.
Fraction A Fraction B Fraction C
K
mG6
(m
M
) 2.65 ± 0.35 0.71 ± 0.07 0.76 ± 0.10
K
iG6
(m
M
) 360 ± 130 31.8 ± 3.1 21.9 ± 2.3
t
1/2
(min) Stable 17 18
N-terminal
sequence
P-E-K-Q-P-L-P-A-A
a
X-Q
b
(S)-Q-P-(I)
c
Properties of CcStP EcMalP EcMalP
a
Residue Ser1 is processed off in CcStP isolated from the natural
organism [15,16].
b
X is an unidentified amino acid.
c

Determin-
ation of the N-terminal sequence of fraction C was not completely
clear at positions 1 and 4.
3324 R. Griessler et al.(Eur. J. Biochem. 271) Ó FEBS 2004
lase was inactive within the limits of detection of the
experimental procedures. Addition of phosphate or phos-
phite restor ed phosphorylase activity, as shown i n
Fig. 5A,B, respectively. The time course of formation of
phosphate was linear w hen phosphate was used as the
activator oxyanion. The c hosen level of phosphate (2.5 or
5m
M
) did not influence the enzymic rate significantly.
When phosphite was the activator oxyanion, the observed
time courses were concave upward, perhaps indicating an
autocatalytic effect of the released phosphate. The reaction
rate recorded at an oxyanion concentration of 2 m
M
was
 4.4 times higher with phosphate than phosphite. Table 4
summarizes the kinetic characterization of PL-CcStP. The
restoration of activity in PL-phosphorylase by phosphate
displayed saturatable concentration dependence, and
half-maximum activation was observed at 0.5 m
M
.At
pH 7.5, about 57% of the wild-type level of activity could be
recovered. The Michaelis constant of the PL-enzyme for
a-
D

-glucose 1-phosphate in the presence of phosphite was
approximately 10 times that of CcStP.
The pH-dependence of activity under conditions of
saturation in both substrates was determined for CcStP
and PL-phosphorylase in the pH range 5.0–8.0. Initial
rates were recorded in t he directions of a-glucan phos-
phorolysis and s ynthesis, and assays for P L-phosphory-
lase in the synthesis direction contained a saturating level
of activating phosphate (2.5 m
M
). Results are shown in
Fig. 6. In either direction of reaction, enzymatic rates
which are effectively turnover numbers (k
cat
) decreased at
high and low p H. Optimum catalytic rates for phos-
phorolysis were found at around pH 7.0 for both the
native enzyme and PL-phosphorylase. In the low pH
region the pH profile of k
cat
for PL-phosphorylase was
displaced outward by  1.0 pH unit, relative to the
corresponding pH profile for CcStP. The decrease in k
cat
(phosphorolysis; k
pho
) a t high p H was similar for both
enzymes. In the synthesis direction, optimum conditions
for k
cat

(k
syn
) w ere observed a t pH 6 .0 for Cc StP. PL-
enzyme bound to phosphate showed maximum activity at
pH 6.5–7.0. The pH profile of k
syn
for PL-phosphorylase
in the presence of phosphate was displaced outward by
 1.0 pH units at high pH, compared to the pH profile of
k
syn
for wild-type CcStP. Fits of the data to Eqn (3)
yielded pK
a
values of 6.9 ± 0.3 and 7.9 ± 0.3 for wild-
type enzyme and PL-CcStP, respectively.
Discussion
Formation and characterization of apo-
Cc
StP
A number of studies have identified prerequisites for
reversible conversion of holo-GP into the a po-enzyme [2]:
localized reversible denaturation promoting subunit disso-
ciation; resolution of PLP through a ldehyde-reactive com-
pounds; and prevention of subunit aggregation. In spite o f
these common characteristics, completely different proto-
cols were ne eded for suc cessful preparation o f apo-enzymes
of RmGP [2], Solanum tuberosum (potato tuber) starch
phosphorylase [11], and EcMalP (D. Palm, unpublished
data). Apo-CcStP was obtained under conditions compar-

able to the ones used by Shaltiel et al. [6] for resolution of
RmGP; i.e. using imidazole citrate and
L
-cysteine as
structure-deforming and PLP-resolving reagents, respect-
ively. Interestingly, however, the pH dependence of the rate
of resolution was opposite in the two e nzymes, CcStP being
stable under the slightly acidic conditions. It was proposed
by others [6–8] that the imidazolium ion is required for
optimum resolution of RmGP at pH  6.0. In Cc StP,
imidazole obviously assists in locally disrupting the native
structure but there was no evidence that its protonated form
would b e p articularly e ffective. M utations within the dimer
contact region of CcStP (Table 2; also [15,18]) had strong
effects on the half-life of activity in resolution buffer.
Likewise, cofactor resolution was inhibited completely
in the presence of phosphate or sulphate. These results are
in good agreement with the notion that weakening
Fig. 5. Restoration of e nzyme a ctivity i n P L-CcStP by exogenous (A)
phosphate a nd (B) phosphite. Incubations w ere carried out a t 30 °Cin
0.1
M
sodium acetate buffe r, pH 7.6, containing  30 lgÆmL
)1
protein.
The substrate levels were constant at 80 gÆL
)1
maltodextrin and 50 m
M
a-

D
-glucose 1-phosphate. T he levels of exogenous activator oxyanion
are indicated by symbols and given in m
M
. In (A) the concentrations of
released phosphate were sufficient to allow an accurate determination
of the a ctivity in spite of the added phosphate. Th e p ossible in hibition
of the enzymatic reaction by phosphate is c ompen sated u sing a high
concentration of a-
D
-glucose 1- phosphate.
Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur. J. Biochem. 271) 3325
subunit-to-subunit interactions in CcStP [15,17,18] is a key
factor driving the resolution of the h olo-enzyme.
Like apo-RmGP, apo-CcStP is monomeric and displays
no enzyme activity. A number of observations indicate that
it retains n ative-like tertiary s tructure. Stabiliz ation of a po-
CcStP by UDP-a-
D
-glucose and ADP-a-
D
-glucose is par-
ticularly relevant because it suggests the preservation of
a cofactor–substrate binding scaffold in apo-CcStP. The
nucleotide-activated sugars structurally resemble the
noncovalent complex of PLP and a-glucose 1-phosphate
that is formed at the phosphorylase active site in the course
of the e nzymatic reaction [20,21]. The available evidence
from gel filtration analysis excludes the occurrence of a
transient apo-dimer lacking phosphorylase activity, induced

by the presence of the stabilizing UDP-a-
D
-glucose. UDP- a-
D
-glucose at a l evel of 5 m
M
inhibits the reaction of n ative
CcStP t o less than 15%, suggesting the absence of a high-
affinity effector site for nucleotide sugars i n the active holo-
phosphorylase dimer. Furthermore, it does not retard the
resolution of the cofactor in CcStP (data not shown),
indicating that the observed stabilizing effect is specific to
the apo-enzyme.
Now, given that PLP resolution caused only minor
denaturation of CcStP tertiary structure, it was especially
interesting that thermostabilization of the holo-enzyme by
phosphate was lost in apo-CcStP; and r ecovered fully upon
reconstitution. This result could indicate that in apo-CcStP
(a) the actual oxyanion b inding site was disrupted, o r (b) a
conformational change t hat accompanies oxyanion b inding
in the holo-enzyme cannot take place. Whatever was truly
responsible, the data suggest that dimerization is required
for restoration of oxyanion-dependent thermostabilization
of CcStP (see below).
Reconstitution of the holo-enzyme
Reconstitution experiments were designed to address two
specific questions of phosphorylase recognition. First, do
apo-phosphorylases of CcStP and EcMalP associate in
solution to form hybrid dimers? Secondly, is there a role of
interactions between protein and oxyanion during the

apofiholo c onversion of CcStP?
Complementation of phosphorylase apo-protomers in
solution has obvious advantages o ver working with immo-
bilized subunits, as described by others [5,7]. However, it
Table 4. Kinetic characterization of PL-CcStP in the presence of activator oxyanion. Initial rates were recorded in 50 m
M
Tris-acetate buffer, pH 7.5,
using a discontinuous assay in which samples were taken after 20, 40 and 60 m in of incubation. The rates were calculated from linear plots of [P
i
]
released against the reaction time. W hen phosphate was the activator oxyanion, initial rates were calculated from t he difference between the
concentrations of total phosphate at a certain in cubation time and phosphate initially pre sent. In all cases this difference w as sufficient to allow
accurate determination of the enzymatic rate. The values of v
max
for the native ph osph orylase determined in the presence and a bsenc e of 10 m
M
phosphite were identical within the experimental e rror, indicating weak (if any) i nhibition by t he added oxyanion. Glc1P, a-
D
-glucose 1-phosphate;
MD, maltodextrin (dextrin equivalent 19.4).
Glc1P (m
M
)orMD(gÆL
)1
) Activator oxyanion (m
M
) v
max
(UÆmg
)1

) K
m
(m
M
)
PL-phosphorylase
1.0–50/80 Phosphite (10) 1.86 ± 0.11 11.2 ± 2.0
50/5–120 Phosphite (10) 1.95 ± 0.12 12.2 ± 2.7
50/80 Phosphate (0.1–3.0) 8.6 ± 0.2 0.49 ± 0.04
Native phosphorylase
1.0–50/80 Phosphite (10) 15.0 ± 0.2 1.08 ± 0.11
Fig. 6. pH profiles i n the direction of a-glucan synthesis (A) an d
phosphorolysis (B) c atalyzed by wild-type CcStP (d) and PL-CcStP (s)
activated by exogenous phosphate ions. (A) Results were ob tained in
0.1
M
sodium ace tate buffer containing 2.5 m
M
P
i
. T he substrate l evels
were 80 gÆL
)1
maltodextrin and 50 m
M
a-
D
-glucose 1-phosphate. Solid
lines are nonlinear fits of the d ata to Eqn (3). For PL-CcStP the cata-
lytic rate at p H 8 was not included in the calculation b ecause i ts value

reflects the effe cts of pH on both rate and enzyme stabilit y. (B) Results
were obtained in 50 m
M
potassium phosphate b uffer containing
80 gÆL
)1
maltodextrin. The li nes indicate t he trend of the d ata.
3326 R. Griessler et al.(Eur. J. Biochem. 271) Ó FEBS 2004
requires m ethods which select f or true hybrids. Mixtures of
reconstituted CcStP and EcMalP were separated by using
hydroxylapatite chromatography [19]. Conditions were
used in which a hybrid would be clearly detectable if it
displayed intermediate binding properties, compared to
wild-type CcStP (weak binding) and EcMalP (strong
binding). The observed elution pattern from t he hydroxyl-
apatite column was not consistent with the formation of
hybrids in substantial amounts. However, a small protein
fraction was detected t hat eluted b efore and after the peaks
clearly assigned to native or reconstituted EcMalP and
CcStP, respectively. This fraction contained e nzyme activity
and obviously, it could b e a phosphorylase hybrid.
Furthermore, we had to consider the possibility that
heterodimers escape detection because the different subunits
interact with hydroxylapatite independently of o ne another.
Therefore, the three protein f ractions obtained (A–C)were
characterized by N-terminal sequencing a nd two parameters
of enzyme function distinguishing sensitively between Cc StP
and EcMalP: (a) apparen t substrate affinity and su bstrate
inhibition in the direction of phosphorolysis of maltodex-
trins; and (b) kinetic s tability at 50 °C. The results showed

that, within limits of detection of the fractionation proce-
dure (5%), only wild-type enzymes were present and no
hybrid dimers formed. The observed small protein peak
(fraction B) very likely contains reconstituted EcMalP, and
its occurrence can be explained by an incomplete retention
of reconstituted Ec MalP by the h ydroxylapatite c olumn. It
seems that t he structural co mplementarity between pro-
tomers of CcStP an d EcMalP was not sufficient for the
different subunits to recognize each other. This finding is
interesting because the packing of hydrophobic r esidues
dispersed over t he main part of the dimer inter face is highly
conserved among known a-glucan phosphorylases [22]
including EcMalP and, by sequence similarity, CcStP. It
suggests that interfacial contacts mediated by polar groups
must be different in EcMalP and CcStP.
We were interested to examine the r elative timing of
steps involved in dimer formation and the appearance of
oxyanion-dependent stabilization of activity during recon-
stitution of apo-CcStP. Analysis of time courses of
recovery of enzyme activity in the absence and presence
of phosphate showed that the yield but not the rate at
which the activity was regained was strongly dependent
on the added phosphate. These observations are novel
and consistent with a mechanism in which the active
dimer is formed first, and enzyme–oxyanion interactions
that are lacking in the monomer are utilized to shift the
equilibrium towards the catalytically c ompetent e nzyme
(Scheme 1). The data are in excellent agreement with the
proposed pathway of thermal denaturation of CcStP [17]
and contribute to an improved understanding of the

effect of phosphate binding on the dimer stability of
CcStP. The evidence presented here and summarized in
Scheme 1 significantly advances the mechanism underly-
ing o xyanion-dependent dimer stabilization because it was
possible for the first time to investigate the properties of
the native-like folded apo-monomer of CcStP. Because
of its low conformational stability under conditions of
thermally induced dissociation of the CcStP subunits, the
apo-monomer usually escaped detection i n the pr evious
studies of CcStP stability [17,18].
Reconstitution of mutant apo-enzymes yielded results
that were fully consistent with recent comparisons of
thermoinactivation rates of the same mutants [15,18].
After correction for differences in protein c oncentration
used, the level of activity r ecovered during the burst ph ase
was s imilar among wild-type and all mutants when no
phosphate was present. Therefore, t his implies that the
mutations did not cause changes in the a ssociation rate of
the phosphorylase subunits. Altered kinetic stab ilities of
the mutants, compared to wild-type, are therefore likely
due to changes in protomer dissociation rate. The effect
of phosphate on the recovery of activity was sensitive to
mutations in the dimer contact region. R234A had lost
much of the apparent affinity of th e wild-type for
phosphate, a nd a phosphate effect on activity recovery
was l acking completely in R242A under t he conditions
used. The data reinforce the conception [15] that the side
chains of Arg234 and Arg242 have key roles in the
mechanism by which phosphate bin ding induces a
kinetically stabilized c onformation of CcStP (Scheme 1).

Restoration of enzyme activity in PL-reconstituted
phosphorylase by exogenous phosphate
The characterization o f CcStP reconstituted w ith PL i n
place of the natural cofactor PLP yielded results that are
relevant in the context of function of the 5¢-phosphate group
in phosphorylase c atalysis [1], as follows. A number of
studies using PL-RmGP have shown that the otherwise
inactive PL-phosphorylase recovered up to 19% of wild-
type activity when exogenous oxyanions were present.
Among a series of compounds tested phosphite was the
most powerful activator anion of PL-RmGP [23,24]. Using
PL-CcStP, phosphate was  4.5-fold more effective than
phosphite, and in saturating concentrations of 3 m
M
it
restored 60% of the original e nzyme activity at pH 7.5. The
data suggest that phosphate binds to the cleft vacated in PL-
CcStP through removal of the original cofactor 5¢-phos-
phate group; and the positions of the dissociable phosphate
in PL-CcStP and the covalently bound phosphate in the
native enzyme are probably similar.
Scheme 1. Formation of active dimers of CcStP during r econstitution of
the apo-phosphorylase with PLP in the absence and presence o f phos-
phate. M is the native-like f olded monomer; M¢ is an irreversibly
denatured monom er; D is the PLP-containing, ac tive d imer; D * is the
stabilized dimer bound to phosphate; M
aggr
is aggregated protei n. All
monomeric forms lack enzyme activity. The denaturation of D as
shown is s up ported by evidence published elsewhere [17].

Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur. J. Biochem. 271) 3327
The direct comparison of pH profiles for the catalytic
rates of CcStP and the complex PL-phosphorylase and
phosphate can arguably provide mechanistic information
because enzyme systems were analyzed whose active sites
differed only by a minimal m odification. However, any
interpretation must be tempered considering that in
RmGP, slightly different binding modes for cofactor-
bound and mobile phosphate groups have been detected
by X-ray crystallography [25]. The question o f interest
was whether differences in pK
a
values for covalent and
noncovalent phosphate (pK
a
¼ 7.2 [23]) groups are
mirrored in the corresponding pH-rate profiles. The
pK
a
values of the cofactor phosphate in unliganded
EcMalP and the EcMalP–arsenate complex are 5.6 [26]
and 6 .7 [27], respectively. The pK
a
of the 5¢-phosphate
group in a model Schiff b ase is 6.2 [ 26]. The available
evidence for EcMalP defines a range of plausible pK
a
values for CcStP because residues interacting with the
5¢-phosphate group in EcMalP are completely conserved
in CcStP. Log k

syn
for the wild-type decreased above an
apparent pK
a
of 6.9 whereas a pK
a
value of 7.9 was
calculated from the pH profile of log k
syn
for PL-CcStP
bound to phosphate. Unfortunately, the activity of PL-
CcStP in the presence of activator phosphite was too low
to permit determination of a reliable pH profile. The
observed DpK
a
of  1.0 pH units would agree reasonably
with DpK
a
¼ 1.2 predicted on the basis of pK
a
values of
phosphate and the cofactor 5¢-phosphate in a model
compound. These data are consistent with a pH-depend-
ent mechanism in which the cofactor phosphate must be
protonated so that catalysis to a-glucan synthesis occurs
[1,28,29]. The pH profiles of log k
pho
for wild-type and
PL-CcStP decreased above an apparent pK
a

value of
 7.3. It is not possible to assign this pK
a
value to the
pH-dependent ionization of a group on the reactive
enzyme–substrate complex; obviously it could reflect the
ionization of the substrate phosphate.
Acknowledgements
The financial support from the Austrian Science Funds (P15118 and
P11898 to B.N.) is gratefully acknowledged. W e t hank Dr Dieter Palm
for communicating a protocol for the p reparation of apo-EcMalP.
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Supplementary material
The following material is available from http://blackwell
publishing.com/products/jou rnals/suppmat/EJB/E JB4265/
EJB4265sm.htm
Fig. S1. Column sizing experiment using Superose 12 HR
10/30 to determine the subunit association state o f CcStP,
apo-CcStP in the absence and presence of 2 mM U DP-a-D-
glucose, and the reconstituted enzyme.
Ó FEBS 2004 Cofactor dissociation studies of starch phosphorylase (Eur. J. Biochem. 271) 3329