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

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

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

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

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

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
+

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
+

. 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

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-

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

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

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

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-

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

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

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

(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-

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

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

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

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).

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

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.
References
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E.J.M. (1990) The role of pyridoxal 5¢-phosphate in glycogen
phosphorylase catalysis. Biochemistry 29, 1099–1107.
2. Graves, D.J. & Wang, J.H. (1972) a-Glucan phosphorylases –
chemical and ph ysical basis of ca talysis a nd regu lation. Ann. Rev.
Biochem. 7, 435–482.
3. Feldmann, K., Zeisel, H.J. & Helmreich, E.J.M. (1976) Com-
plementation of subunits from glycogen pho sphorylases of frog
and rabbit skeletal muscle and rabbit liver. Eur. J. Biochem. 65,
285–291.
4. Tu, J I. & Graves, D.J. (1973) Association-dissociation properties
of sodium borohydride -reduce d phosphorylase b. J. Biol. Chem.
248, 4617–4622.
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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