Báo cáo khoa học: The phosphate site of trehalose phosphorylase from Schizophyllum communeprobed by site-directed mutagenesis and chemical rescue studies pptx

The phosphate site of trehalose phosphorylase from
Schizophyllum commune probed by site-directed
mutagenesis and chemical rescue studies
Christiane Goedl and Bernd Nidetzky
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
Glycosyltransferases (GTs) constitute a diverse class of
enzymes that catalyze the synthesis of glycosidic bonds
in oligosaccharides and glycoconjugates. A nucleotide-,
phospho- or lipid-phospho-activated sugar is typically
utilized as the donor substrate, and transfer of the gly-
cosyl moiety to the acceptor molecule occurs with
either inversion or retention of configuration at the
reactive anomeric carbon [1]. After detailed studies of
glycogen phosphorylase spanning many decades [2–6],
there has been recent rekindled interest in the mecha-
nistic characterization of retaining glycosyltransferases,
particularly in relation to glycoside hydrolases, the
physiological counterpart enzymes that catalyze the
breakdown of glycosidic linkages [7]. The canonical
Keywords
catalytic mechanism; chemical rescue;
family GT-4; glycosyltransferase; a-retaining
glucosyl transfer
Correspondence
B. Nidetzky, Institute of Biotechnology and
Biochemical Engineering, Graz University of
Technology, Petersgasse 12 ⁄ I, 8010 Graz,
Austria
Fax: +43 316 873 8434
Tel: +43 316 873 8400
E-mail:

(Received 5 October 2007, revised 10
December 2007, accepted 19 December
2007)
doi:10.1111/j.1742-4658.2007.06254.x
Schizophyllum commune a,a-trehalose phosphorylase utilizes a glycosyl-
transferase-like catalytic mechanism to convert its disaccharide substrate
into a-d-glucose 1-phosphate and a-d-glucose. Recruitment of phosphate
by the free enzyme induces a,a-trehalose binding recognition and promotes
the catalytic steps. Like the structurally related glycogen phosphorylase
and other retaining glycosyltransferases of fold family GT-B, the trehalose
phosphorylase contains an Arg507-XXXX-Lys512 consensus motif (where
X is any amino acid) comprising key residues of its putative phosphate-
binding sub-site. Loss of wild-type catalytic efficiency for reaction with
phosphate (k
cat
⁄ K
m
=21000m
)1
Æs
)1
) was dramatic (‡10
7
-fold) in purified
Arg507 fi Ala (R507A) and Lys512 fi Ala (K512A) enzymes, reflecting a
corresponding change of comparable magnitude in k
cat
(Arg507) and K
m
(Lys512). External amine and guanidine derivatives selectively enhanced

the activity of the K512A mutant and the R507A mutant respectively.
Analysis of the pH dependence of chemical rescue of the K512A mutant
by propargylamine suggested that unprotonated amine in combination with
H
2
PO
4
)
, the protonic form of phosphate presumably utilized in enzymatic
catalysis, caused restoration of activity. Transition state-like inhibition of
the wild-type enzyme A by vanadate in combination with a,a-trehalose
(K
i
= 0.4 lm) was completely disrupted in the R507A mutant but only
weakened in the K512A mutant (K
i
= 300 lm). Phosphate (50 mm) enhan-
ced the basal hydrolase activity of the K512A mutant toward a,a-trehalose
by 60% but caused its total suppression in wild-type and R507A enzymes.
The results portray differential roles for the side chains of Lys512 and
Arg507 in trehalose phosphorylase catalysis, reactant state binding of
phosphate and selective stabilization of the transition state respectively.
Abbreviations
G1P, a-
D-glucose 1-phosphate; GTs, glycosyltransferases; K512A, Lys512 fi Ala mutant; R507A, Arg507 fi Ala mutant; ScTPase,
Schizophyllum commune trehalose phosphorylase; S
N
i-like, internal return-like mechanism.
FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 903
mechanism of a retaining glycoside hydrolase is that of

a double-displacement reaction involving a covalent
glycosyl-enzyme intermediate [4,8–10]. Because evi-
dence from structural and mutagenesis studies has so
far failed to support a similar type of intermediate for
retaining glycosyltransferases [6,7,11–14], an alternative
mechanistic scenario termed internal return-like (S
N
i-
like) was considered in which hypothetically, direct
front-side displacement of the leaving group by the
incoming nucleophile would result in retention of the
anomeric configuration (Fig. 1). Reaction is proposed
to occur via a single transition state featuring a highly
developed oxocarbenium-ion character. A strict
requirement for the tentative S
N
i-like mechanism is
that donor and acceptor substrates are precisely posi-
tioned in close proximity to each other in the enzyme
active site. Indeed, reaction through a ternary complex
where both substrates must bind to the enzyme before
the first product is released appears to be a defining
catalytic feature of retaining glycosyltransferases;
structural insights into enzymatic glycosyl transfer via
a ternary complex are provided elsewhere [3,5,12,15].
However, little is known about catalytic factors that
could facilitate enzymatic glycosyl transfer via the pro-
posed S
N
i-like process, and the stabilization of the

oxocarbenium ion-like species in the transition state.
The present study was concerned with the quantitative
analysis of the role of noncovalent interactions
between active-site residues of the enzyme and the
phosphate nucleophile ⁄ leaving group in a reaction cat-
alyzed by a sugar 1-phosphate dependant transferase.
Schizophyllum commune trehalose phosphorylase
(ScTPase; EC 2.4.1.231) utilizes a glycosyltransferase-
like catalytic mechanism to convert a,a-trehalose and
phosphate into a-d-glucose 1-phosphate (G1P) and
a-d-glucose in a freely reversible reaction [16,17]. In
the direction of phosphorolysis, recruitment of phos-
phate by the free enzyme induces binding recognition
for a,a-trehalose and promotes the catalytic steps of
glucosyl transfer. d-glucose is released from the ternary
enzyme–product complex, and dissociation of G1P
regenerates the free enzyme [17]. The unreactive phos-
phate-analogue vanadate is a transition state-like
inhibitor of ScTPase [18,19] whereby partial mimicry
of the transition state was proposed to derive from a
hydrogen bond between vanadate and the a-anomeric
hydroxyl of the glucose leaving group ⁄ nucleophile
(Fig. 1). Unlike glycogen phosphorylase, which utilizes
pyridoxal 5¢-phosphate to promote the attack of the
phosphate [5] and other nucleotide sugar-dependent
glycosyltransferases, which often require a metal ion
for activation of the leaving group [20–22], ScTPase
does not employ a cofactor in catalysis.
Based on sequence similarity, ScTPase has been clas-
sified into family (GT)-4 of the glycosyltransferase fam-

ilies. Recent crystal structures of three representatives
of family GT-4 [23,24] revealed a common protein
structural organization typical of transferases of fold
family GT-B where the catalytic centre, which features
a highly conserved architecture, is situated in a deep
cleft formed by two Rossman-fold domains. The struc-
ture of Mycobacterium smegmatis phosphatidylinositol-
mannosyltransferase (PimA) in complex with the natural
sugar-donor substrate GDP-mannose showed that the
distal phosphate moiety of the GDP leaving group was
tightly coordinated by strong hydrogen bonds with
Gly16, Arg196 and Lys202 [23]. The suggestions from
a mutational analysis of ScTPase that the homologous
Gly292, Arg507 and Lys512 (see supplementary
Fig. S1) serve a key role in the phosphorylase reaction
as phosphate-binding residues are thus strongly sup-
ported [16]. Numbering of ScTPase starts with the
initiator methionine as 1 and does not consider the
N-terminal 11 amino acid-long fusion peptide that is
used for recombinant protein production. In the pres-
ent study, we have extended significantly the previous
Fig. 1. Reaction of trehalose phosphorylase
via an S
N
i-like mechanism proposed for
retaining glycosyltransferases where direct
front-side displacement results in retention
of anomeric configuration. A predicted gen-
eral feature of the mechanism is the devel-
opment of a strong hydrogen bond between

the incoming nucleophile and the leaving
group in the transition state of the reaction.
The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky
904 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS
scanning mutagenesis of the active site of trehalose
phosphorylase [16] and report on results of steady-
state kinetic analysis and chemical rescue studies for
site-directed ScTPase mutants Arg507 fi Ala (R507A)
and Lys512 fi Ala (K512A). A detailed portrait of the
catalytic function of the two basic side chains is pro-
vided. These new insights are of general interest con-
sidering the presence of an active-site consensus motif,
Arg507-XXXX-Lys512 (where X is any amino acid) as
in ScTPase, in glycogen phosphorylase [3,6] and other
retaining clan IV glycosyltransferases of fold family
GT-B [12,23,25]. Interestingly, mammalian and yeast
glycogen synthases of family GT-3 appear to have
replaced the Arg-XXXX-Lys motif of their bacterial
and plant counterpart enzymes in family GT-5 by two
conserved clusters of Arg residues [26,27]. Likewise,
GT-A fold glycosyltransferases also utilize a conserved
diad of Arg and Lys for binding of the donor substrate
pyrophosphate group and in catalysis. The two basic
residues occur in a motif displaying the inverted GT-B
pattern, Lys359-XXXXX-Arg365 as in bovine a-1,3-
galactosyltransferase of family GT-6 [28]. Despite the
evidence provided by the high-resolution crystal struc-
tures for many of these glycosyltransferases, the role
of the conserved residues for promoting a-retaining
glycosyl transfer has not been well defined using muta-

genesis and detailed kinetic analysis.
Results
Analysis of kinetic consequences in R507A and
K512A mutants of ScTPase
CD spectra of purified wild-type and mutant trehalose
phosphorylases were almost superimposable on each
other (see supplementary Fig. S2), indicating that the
relative proportion of secondary structural elements in
the folded structure of the wild-type enzyme was not
altered significantly in R507A and K512A mutants.
Therefore, this strongly suggests that kinetic conse-
quences resulting from the replacement Arg507 fi Ala
or Lys512 fi Ala are not due to partial misfolding of
the mutant enzymes. Figure 2 displays results of the
steady-state kinetic characterization of R507A and
K512A, and kinetic parameters of wild-type and
mutant phosphorylases for phosphorolysis of a,a-tre-
halose are summarized in Table 1. Both R507A and
K512A exhibited a dramatic ( ‡10
7
-fold) loss of cata-
lytic efficiency for reaction with phosphate (k
cat
⁄ K
m
)
in comparison with the wild-type enzyme. In R507A,
the effect on k
cat
⁄ K

m
was distributed between a major,
4.1 · 10
5
-fold decrease in apparent catalytic centre
activity (k
cat
) and a comparably minor, 130-fold
increase in the value of K
m
for phosphate. In K512A,
by contrast, the initial phosphorolysis rate was linearly
dependent on the concentration of phosphate up to
1.5 m (Fig. 2), precluding determination of k
cat
and K
m
Fig. 2. Steady-state kinetic characterization of R507A (s) and
K512A (d) mutant trehalose phosphorylases. Reaction rates (V)
were recorded in 50 m
M Mes buffer, pH 6.6, using a constant con-
centration of 400 m
M a,a-trehalose. [E] is the molar enzyme con-
centration. Reaction mixtures were incubated at 30 °C for up to
40 h, and the release of G1P was determined enzymatically. A plot
of concentration of G1P released against the incubation time was
linear in all cases, allowing determination of V and showing that
both enzymes were reasonably stable under the incubation condi-
tions. Error bars indicate the SD of four independent determina-
tions.

Table 1. Comparison of kinetic parameters for wild-type, R507A and K512A mutant trehalose phosphorylases in a,a-trehalose phosphoroly-
sis direction at 30 °C and pH 6.6.
k
cat
⁄ K
m phosphate
[10
)3
ÆM
)1
Æs
)1
]
Fold
decrease
K
m phosphate
[mM]
Fold
increase
K
ic vanadate
[lM]
Fold
increase
V
hydrolysis
⁄ [E] [10
)4
Æs

)1
]
0m
M
phosphate
a
50 mM
phosphate
ScTPase 2.1 · 10
7
± 0.4 · 10
7
1 0.8 ± 0.1 1 0.4 ± 0.1 1 3.5 ± 0.4 No hydrolysis
R507A 0.4 ± 0.1 5.3 · 10
7
103 ± 32 129 No inhibition 5.0 ± 0.6 No hydrolysis
K512A 1.4 ± 0.2 1.5 · 10
7
No saturation 298 ± 51 745 15 ± 1.2 24 ± 2.3
a
Data from [16].
C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase
FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 905
as independent kinetic parameters. The value of
k
cat
⁄ K
m
was obtained from the slope of the straight
line fitted to the data in Fig. 2. It was 1.5 · 10

7
-fold
lower than the corresponding catalytic efficiency of the
wild-type enzyme. The decrease in k
cat
⁄ K
m
for K512A
must therefore reflect a very large (>2000-fold)
increase in K
m
, compared with the wild-type values,
suggesting that the site-directed replacement of Lys512
caused a more substantial disruption of binding affin-
ity for phosphate than that of Arg507.
Table 1 also summarizes values of K
ic vanadate
for
wild-type and mutant trehalose phosphorylases. As in
the wild-type enzyme, vanadate acted as a competitive
inhibitor against phosphate in K512A. However,
K
ic vanadate
was increased 745-fold as result of the site-
directed replacement Lys512 fi Ala. By contrast,
R507A was not at all inhibited by the used concentra-
tions of vanadate (0.5–5.0 mm). The ratio of
K
m
⁄ K

ic vanadate
was therefore changed as result of the
site-directed substitution of Arg507 from a value of
2000 in the wild-type enzyme to an infinitesimally
small value (=103 ⁄¥) in the mutant. It appears to
have been increased to a value significantly >2000
(=1600 ⁄ 0.3) in K512A.
The hydrolase activities of wild-type and mutant tre-
halose phosphorylases towards a,a-trehalose were com-
pared under hydrolysis-only conditions [16] and under
conditions where, in the presence of 50 mm of phos-
phate, phosphorolysis competed with hydrolysis of the
disaccharide. The results are summarized in Table 1.
Inhibition by vanadate of the hydrolysis of a,a-treha-
lose and G1P catalyzed by wild-type ScTPase was also
measured. With the methods used, it was not possible
to quantify, in the wild-type, a small proportion of
a,a-trehalose conversion by ‘error hydrolysis’ next to
an overwhelmingly predominant phosphorolysis reac-
tion, which also produces d-glucose. However, within
limits of detection of the experimental procedures
(£1%), no hydrolysis of a,a-trehalose by wild-type
enzyme took place when 50 mm of phosphate was
present. Because replacement of Arg507 caused selec-
tive slowing down of the phosphorolysis reaction com-
pared with the hydrolysis of a,a-trehalose [16], the
complete suppression of the hydrolase activity of
R507A towards a,a-trehalose upon addition of 50 mm
of phosphate could be established unambiguously. By
marked contrast, the basal rate of hydrolysis of

a,a-trehalose by K512A was enhanced significantly
(approximately 1.6-fold) in the presence of 50 mm of
phosphate. By contrast to the clear inhibitory effect of
vanadate on the hydrolysis of a,a-trehalose by wild-
type ScTPase (3.5-fold), vanadate did not inhibit the
hydrolysis of G1P by the same enzyme. Values of
V
hydrolysis
⁄ [E] were 4.0 · 10
)4
Æs
)1
and 3.9 · 10
)4
Æs
)1
in
the absence and presence of vanadate, respectively.
Noncovalent complementation of trehalose
phosphorylase activity in R507A and K512A
Inclusion of 200 mm of guanidine into the assay for
phosphorolysis of a,a-trehalose at pH 6.6 caused
45-fold enhancement of the activity of R507A seen
in the absence of guanidine (k
0
= 4.2 · 10
)5
Æs
)1
).

Likewise, a 23-fold stimulation of the basal activity
of K512A (k
0
= 6.1 · 10
)5
Æs
)1
) was observed in the
presence of 200 mm of propargylamine. Functional
complementation of R507A and K512A, expressed as
k
rescue
⁄ k
0
, displayed a hyperbolic dependence on the
concentration of the respective rescue reagent (Fig. 3).
Values of k
max
(R507A: 2.8 · 10
)3
Æs
)1
; K512A:
1.6 · 10
)3
Æs
)1
) and K
R
(guanidine, 100 mm; propargyl-

amine, 58 mm) were obtained with a relative SD of
approximately 6% and 10%, respectively, using non-
linear fits of Eqn (1) to initial-rate data recorded in
the absence and presence of rescue reagent concentra-
tions in the range 10–200 mm. Guanidine and propar-
gylamine did not exhibit a significant effect on the
activity of the wild-type enzyme, except for a weak
inhibition (<50% reduction in rate) by concentrations
of guanidine higher than 100 mm. No cross-reactiva-
tion of R507A by propargylamine (10–200 mm) and
K512A by guanidine (10–200 mm) was observed. Addi-
tion of 200 mm of NaCl did not alter the activity of
either one of the site-directed mutants. These results
Fig. 3. Functional complementation of mutant trehalose phosphory-
lases. R507A was reactivated by guanidine (s) and K512A by prop-
argylamine (d). No cross-reaction was observed, and the rescue
agents did not significantly alter the wild-type activity (guanidine ,,
propargylamine
). Lines show the fit of Eqn (1) to the data.
The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky
906 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS
provide strong evidence against the possibility of a non-
specific activation of ScTPase mutants and suggest that
external guanidine and propargylamine can partly com-
pensate for the loss of the original side chain in R507A
and K512A, respectively.
A series of primary amines and derivatives of guani-
dine were therefore examined for their ability to restore
phosphorylase activity in K512A and R507A, respec-
tively. The results obtained are summarized in Table 2,

along with relevant structural and electronic parameters
of the compounds used for chemical rescue. Because
k
rescue
for R507A and K512A appeared to exhibit a
complex dependence on steric factors of the rescue
reagents and the set of amine and guanidine compounds
tested was rather small (five each), we did not pursue
construction of the respective Brønsted plot using quan-
titative structure–activity relationship analysis. How-
ever, even in the absence of correction for the influence
of molecular volume and hydrophobicity of the rescue
reagent, Table 2 shows clearly that the effect of the pK
a
of the amine and guanidine derivatives on specific resto-
ration of activity in K512A and R507A, respectively,
was very small and probably not significant.
Analysis of the pH dependence of functional
complementation of K512A
k
max
⁄ K
R
for chemical rescue of K512A by propargyl-
amine and R507A by guanidine was pH-dependent.
Its value decreased in K512A from 0.035 m
)1
Æs
)1
at

pH 6.6 to 0.003 m
)1
Æs
)1
at pH 8.2, and in R507A
from 0.028 m
)1
Æs
)1
at pH 6.6 to 0.005 m
)1
Æs
)1
at
pH 8.0. These pH effects are explicable on account
of changes in the ionization states of the enzyme
and the substrate phosphate (pK
a,2
= 7.2) and, in
the case of K512A, deprotonation of propargylamine
at high pH (pK
a
= 8.2). Analysis of pH-rate profiles
for wild-type ScTPase suggested that H
2
PO
4
)
is the
protonic form of phosphate utilized in the enzymatic

reaction [17]. The pH-dependence of functional com-
plementation of K512A was therefore examined in
more detail.
Figure 4 compares pH-rate profiles of K512A
assayed in the absence and presence of 200 mm of
propargylamine with the corresponding pH-rate pro-
file of the wild-type enzyme (Fig. 4A) and summarizes
the results of chemical rescue experiments with
K512A carried out at four different pH values
(Fig. 4B). Figure 4A shows that pH-rate profiles of
wild-type enzyme and K512A were similar, both
showing maximum enzyme activity at an approximate
pH of 6.5. Addition of propargylamine caused an up-
shift of the optimum pH of K512A by approximately
1 pH unit. Restoration of trehalose phosphorylase
activity in K512A by propargylamine was best at
pH 7.5 where a value of 140 was observed for
k
rescue
⁄ k
0
when the concentration of rescue reagent
was saturating (Fig. 4B). The presence of 100 mm of
propargylamine caused an approximately 10-fold
enhancement of the catalytic efficiency of K512A for
reaction with phosphate at pH 6.6, in reasonable
agreement with the results obtained in activity assays
at a single phosphate concentration of 50 mm
(Table 2). Likewise, under conditions of chemical res-
cue of K512A by 200 mm of propargylamine, k

cat
⁄ K
m
for phosphate increased from a value of 0.014 m
)1
Æs
)1
at pH 6.6 to 0.023 m
)1
Æs
)1
at pH 7.5, suggesting
that the corresponding pH-rate profile in Fig. 4A
reflects the pH dependence of k
cat
⁄ K
m
. These results
indicate that, for optimum restoration of activity in
K512A, the protonation states of propargylamine and
phosphate must be matched. We found that there was
a good linear correlation between log(k
rescue
⁄ k
0
) and
the limiting concentration of either one of the
Table 2. Chemical rescue analysis for R507A and K512A. The concentration of external reagent was 200 mM. Values of V ⁄ [E] were
recorded at 30 °Cin50m
M Mes buffer, pH 6.6, using 400 mM a,a-trehalose and 50 mM potassium phosphate as substrates. Molecular

volume (Mol. volume) and hydrophobicity (logP) were calculated using the programs
SPARTAN 06, version 1.1.0 and KOWWIN, respectively.
pK
a
values are from the literature [32,39].
R507A pK
a
Molecular
volume [A
˚
3
] logP
V ⁄ [E]
[10
)4
Æs
)1
]
a
Fold
increase
a
K512A pK
a
Molecular
volume [A
˚
3
] logP
V ⁄ [E]

[10
)4
Æs
)1
]
a
Fold
increase
a
No additive 0.42 1 No additive 0.61 1
Guanidine 13.6 59.1 )1.63 19 45 Methylamine 10.6 46.5 )0.64 10 16
Methylguanidine 13.4 79.5 )1.16 5.2 12 Ethylamine 10.6 65.0 )0.15 24 39
Ethylguanidine 13.3 98.1 )0.67 5.4 13 Ammonia 9.2 25.4 )1.38 0.9 1.5
Acetamidine 12.5 67.7 )2.52 2.0 4.8 Propargylamine 8.2 75.8 )0.43 14 23 (24)
b
Aminoguanidine 11.0 71.3 )1.99 1.6 3.8 2,2,2-Trifluoro
ethylamine
5.7 79.5 0.27 6.7 11 (122)
b
a
V ⁄ [E] measured in the absence and presence of rescue reagent are referred to as k
0
and k
rescue
in text, respectively. Likewise, k
rescue
⁄ k
0
in text corresponds to fold increase.
b

Values in parentheses were corrected for the fraction of protonated amine.
C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase
FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 907
compounds, H
2
PO
4
)
and unprotonated amine, in this
combination of protonic forms (Fig. 4B, inset).
Discussion
Evidence from high-resolution X-ray structures
[3,12,25,29] and site-directed mutagenesis [13,30,31]
of glycosyltransferases of fold family GT-B supports
an important role for the consensus motif, Arg507-
XXXX-Lys512 as in ScTPase, in binding recognition
of the phospho leaving group of the glycosyl donor
substrate. However, little is known about the individ-
ual contribution of each side chain to catalytic
efficiency. The present study of Sc TPase used site-
directed replacement by Ala and detailed kinetic
comparison of wild-type and mutant enzymes to
portray the tasks fulfilled by Arg507 and Lys512 in
the interaction network of active site residues during
binding of phosphate and in catalysis. Although we
consider a-retaining glucosyl transfer via the S
N
i-like
mechanism plausible for ScTPase (Fig. 1), the results
presented here do not provide evidence that would

settle the mechanistic debate surrounding this and
other retaining glycosyltransferases.
Proposed roles for Arg507 and Lys512 in the
mechanism of ScTPase deduced from analysis
of kinetic consequences of their individual
replacements by Ala
The steady-state ordered kinetic mechanism of ScTPase
where phosphate binds before a,a-trehalose [17] implies
that k
cat
⁄ K
m
for phosphate is a second-order rate con-
stant for the association between the free phosphory-
lase and the nucleophile of the reaction. k
cat
is thought
to measure the rate-determining conversion of the ter-
nary enzyme–substrate complex [17]. Individual
replacements of Arg507 and Lys512 caused disruption
of the phosphate binding rate by more than seven
orders of magnitude, which is equivalent to an ener-
getic destabilization of ‡41 kJÆmol
)1
(=RT · ln10
7
where R is the gas constant and T is a temperature of
303.15 °K), compared with the wild-type enzyme. Equi-
librium binding in terms of K
m

for phosphate appeared
to be completely destroyed in K512A whereas it was
weakened in a comparatively moderate way (130-fold)
in R507A. Interestingly, relevant single-site mutants
of family GT-35 maltodextrin phosphorylase
(Arg535 fi Gln; Lys540 fi Arg) [31] and family GT-5
glycogen synthase (Arg300 fi Ala; Lys305 fi Ala) [13],
both from Escherichia coli , showed closely similar
K
m
values to their wild-type forms. Their catalytic cen-
tre activities, however, were between three to four
orders of magnitude below the corresponding wild-type
levels. In k
cat
terms, the dimension of loss of catalytic
activity was significantly higher in R507A than the
comparable Arg mutants of the two other transferases.
Noteworthy, a Lys211 fi Ala mutant of Acetobacter
xylinum a-mannosyltransferase, which shares with
ScTPase the membership to family GT-4, was reported
to be devoid of any enzyme activity [30]. The crystallo-
graphically determined hydrogen bond distance
between oxygens of the distal phospho group of UDP-
glucose and the side chains of Arg196 and Lys202 of
family GT-4 mannosyltransferase PimA was only 2.44
and 2.77 A
˚
, respectively [23]. Removal of either one of
the two strong bonds by mutagenesis would therefore

be expected to result in marked loss of the binding
energy used by the wild-type enzyme to promote the
reaction. Kinetic consequences for R507A and K512A
mutants of ScTPase are consistent with this structure-
derived suggestion.
Fig. 4. pH-dependence of functional complementation of K512A by propargylamine. Reaction rates were recorded using 50 mM potassium
phosphate and 400 m
M a,a-trehalose. (A) pH profiles for wild-type enzyme (d), K512A (s) and K512A determined in the presence of
200 m
M propargylamine (.). (B) Chemical rescue of K512A at pH 6.6 (d), 7.5 (s), 8.2 (.) and 8.5 (n). The inset displays the logarithmic
dependence of k
rescue
⁄ k
0
on the relative content of active compound (H
2
PO
4
)
and NH
2
) present.
The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky
908 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS
The comparison of apparent affinities for reversible
binding of phosphate (K
m
) and the transition state
mimic vanadate (K
i

) by wild-type and mutant forms of
trehalose phosphorylase (Table 1) delineates differen-
tial roles for Lys512 and Arg507 in the enzymatic
mechanism. The particular change in the ratio of
K
m
⁄ K
i
resulting from site-directed substitution of
Lys512 (increase) and Arg507 (decrease) compared
with the wild-type value suggests that, whereas Lys512
appears to be primarily required for phosphate binding
in the reactant state, Arg507 promotes the catalytic
step of glucosyl transfer through a selective stabiliza-
tion of the transition state of the reaction. Occupancy
of the phosphate binding site in wild-type and R507A
phosphorylases caused complete shut-down of their
hydrolytic activity towards a,a-trehalose in the absence
of phosphate whereas, in K512A, addition of phos-
phate stimulated weakly the breakdown of the disac-
charide via hydrolysis. Steps involved in phosphate
binding by the wild-type enzyme arguably include an
obligatory exclusion of water from the catalytic site.
Their drastic impairment resulting from the site-direc-
ted substitution of Lys512 is likely to be responsible
for this unusual property of the K512A mutant.
Interpretation of results of functional
complementation studies
Chemical rescue experiments, in which a small mole-
cule compensates for the missing side chain of a rele-

vant site-directed mutant, often provide valuable
insights into the role of active-site arginine [32–36] and
lysine residues [37–40] for the catalytic function of dif-
ferent enzymes. In the present study, we show that
activity lost in Arg507 fi Ala and Lys512 fi Ala vari-
ants of ScTPase could be selectively restored by deriv-
atives of guanidine and primary amines, respectively.
The failure of amines to rescue R507A and, likewise,
guanidine derivatives to rescue K512A is consistent
with observations made with relevant mutants of sev-
eral other enzymes [32–36], and it also supports the
notion that Arg507 and Lys512 fulfill different tasks in
trehalose phosphorylase catalysis (see above).
Partial functional complementation of the catalytic
defect in R507A by guanidine displayed saturation
behavior with respect to both the rescue agent and
the substrate. Therefore, this suggests that guanidine
binds to the cleft vacated by the replacement of the
side chain of Arg507 in the mutant and both the
rescue agent and the substrate phosphate form a ter-
nary complex prior to catalysis. Considering a pK
a
for guanidine of approximately 13.6, we conclude
from analysis of the pH dependence of the second-
order rate constant for the chemical rescue process
that the protonated guanidinium ion is most likely
required for noncovalent restoration of phosphory-
lase activity in R507A. The observed 5.6-fold dec-
rease in k
max

⁄ K
R
in response to an increase in pH
from 6.6 to 8.0 would be readily explained by depro-
tonation of the phosphate monoanion, which is the
form of the substrate presumably utilized in the
enzymatic reaction [17], and parallels the effect of
the same pH change on k
cat
⁄ K
m
for phosphate in
wild-type ScTPase.
Chemical rescue of K512A by propargylamine
exhibited a complex pH dependence, likely explicable
on account of the similar pK
a
values for the external
reagent (pK
a
= 8.2) and the substrate phosphate
(pK
a,2
= 7.2). However, the observed pH effects on
k
max
⁄ K
R
for propargylamine and k
cat

⁄ K
m
for phos-
phate determined in the absence and presence of a sat-
urating concentration of propargylamine (200 mm;
4 · K
R
at pH 6.6) would be best explained if unproto-
nated propargylamine and H
2
PO
4
)
were involved in
the catalytic reaction of an optimally rescued K512A
mutant. The scenario proposed for propargylamine
need not be the same for methylamine and ethylamine,
which, in spite of their high pK
a
of 10.6, exhibit
comparable efficiency to propargylamine as a rescue
reagent of K512A at pH 6.6 (Table 2). Binding of
propargylamine to K512A failed to restore, in terms of
the K
m
value, some of the affinity of wild-type treha-
lose phosphorylase for phosphate. Therefore, to what
extent the function of the original side chain of Lys512
can be gauged by the results of our chemical rescue
studies remains elusive.

We plotted log(k
rescue
⁄ k
0
) of R507A and K512A
against the pK
a
of the rescue agent taking data from
Table 2, assuming that all of the listed derivatives of
guanidine and primary amines fit the respective cavity
resulting from the replacement of the side chain of
Arg507 (98 A
˚
3
) and Lys512 (80 A
˚
3
) by the side chain
of Ala (19 A
˚
3
). These limited Brønsted plots did not
detect a significant correlation between rescue efficacy
and reagent pK
a
and therefore do not support a role
for Arg507 and Lys512 in catalytic proton transfer by
ScTPase. However, in the phosphorolysis direction of
the enzymatic reaction (Fig. 1), partial protonation of
the glycosidic oxygen of a,a-trehalose will be needed

to facilitate the departure of the leaving group. In the
hypothetical S
N
i-like catalytic mechanism of the treha-
lose phosphorylase, enzyme-bound H
2
PO
4
)
is a strong
candidate to fulfill the role of the proton donor. The
side chains of Arg507 and Lys512 could provide assis-
tance in this process via electrostatic stabilization and
positioning of the phosphate ligand.
C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase
FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 909
Because of the similarity in the reactions catalyzed,
it is interesting to compare trehalose phosphorylase
with a-1,4-glucan phosphorylase. Crystal structures of
E. coli maltodextrin phosphorylase bound with phos-
phate and oligosaccharide show that hydrogen bonds
from the NE and NH2 atoms of the guanidine side
chain of Arg535 to two oxygen atoms of phosphate
dominate the contacts between the enzyme and the
phosphate group [3,6]. The side chain of Lys540 and
the main chain N of Gly115 (corresponding to Gly292
in ScTPase) also interact with the phosphate ligand,
which is brought into a plausible catalytic position
within hydrogen-bonding distance of the reactive gly-
cosidic oxygen of the oligosaccharide. Assuming that a

similar network of protein contacts positions vanadate
at the active site of ScTPase, mutation of the key
Arg507 into Ala would be expected to disrupt the pro-
posed hydrogen bond between vanadate and the glyco-
sidic oxygen of a,a-trehalose, consistent with the
observed complete loss of transition state-like inhibi-
tion by vanadate in R507A. In maltodextrin phosphor-
ylase, the side chain of Lys540 also hydrogen bonds
with the 5¢-phosphate moiety of the pyridoxal
phosphate cofactor. These contacts stabilize the cata-
lytic 5¢-phosphate group in a position within hydrogen
bonding distance of the substrate phosphate from
which the attack of inorganic phosphate on the glyco-
sidic oxygen is promoted. Furthermore, the highly con-
served Glu638 (Glu606 in ScTPase), which is also
located at the sugar–phosphate contact region, forms a
salt bridge with Lys540. Results from
31
P-NMR stud-
ies revealed an indirect interaction of Glu638 with the
5¢-phosphate group of the cofactor [41] and suggested
participation of the glutamate in establishing a catalyt-
ically relevant network of charged groups in the active
site [42]. However, the absence of pyridoxal phosphate
in ScTPase implies that the role of the conserved lysine
in promoting the enzymatic reaction need not be
identical for the two phosphorylases.
In summary, based on the evidence obtained in
the present study, we propose differential roles for
the side chains of Lys512 and Arg507 in trehalose

phosphorylase catalysis. Although Lys512 is required
for binding of the phosphate nucleophile in the reac-
tant state, Arg507 facilitates the reaction through a
selective stabilization of the transition state. In the
proposed S
N
i-like mechanism of ScTPase (Fig. 1),
electrostatic ‘front-side’ stabilization of the oxocarbe-
nium ion-like transition state by the incoming
phosphate nucleophile could be a decisive catalytic
factor. Arg507 might contribute indirectly to this sta-
bilization by bringing the phosphate into a suitable
position.
Experimental procedures
Materials and enzymes
Unless otherwise noted, all materials used have been
described elsewhere [17,43]. Purified preparations of wild-
type ScTPase as well as R507A and K512A mutants
thereof were obtained using previously reported procedures
[16]. Enzyme stock solutions containing approximately
5 mg proteinÆmL
)1
were stored in 50 mm potassium–phos-
phate buffer, pH 7.0, and kept at )21 ° C until use.
Protein characterization
Thawed protein samples were checked by SDS ⁄ PAGE to
ensure that partial N-terminal truncation of the phosphor-
ylase preparations [16] had not occurred during storage.
Far-UV CD spectra of wild-type and mutant phosphory-
lases were acquired at 30 °C with a J-715 spectropolari-

meter (Jasco Inc., Easton, MD, USA) using a 0.1-cm
path length cylindrical cell and instrument settings: step
resolution = 0.2 nm; scan speed = 50 nmÆmin
)1
; response
time = 1 s; bandwidth = 1 nm. Triplicate spectra were
recorded in the wavelength range 260–190 nm using
enzymes (approximately 1.6 mgÆmL
)1
) dissolved in 50 mm
potassium–phosphate buffer, pH 7.0. They were subse-
quently averaged and corrected by a blank spectrum lack-
ing enzyme. Smoothing and normalizing was performed
using a molecular mass of 82.8 kDa for full-length
ScTPase. Protein concentration was determined using the
Bio-Rad dye-binding method (Bio-Rad, Vienna, Austria)
referenced against BSA as the standard. We are unaware
of a method for the titration of active sites in prepara-
tions of trehalose phosphorylase. Therefore, the molar
enzyme concentration [E] was calculated, in a commonly
used procedure, from the concentration of purified pro-
tein. Because all enzyme preparations were obtained and
treated in exactly the same way and displayed similar sta-
bilities of their activities during storage (data not shown),
values of [E] for wild-type and mutant enzymes are
without internal bias.
Steady-state kinetic characterization
Buffer exchange to 50 mm Mes, pH 6.6–7.5, and
50 mm Tes, pH 7.5–8.5, was achieved through repeated
ultrafiltration of protein stock solutions using 10-kDa

cut-off Vivaspin 500 microconcentrator tubes (Sartorius,
Gottingen, Germany). Initial rates of phosphorolysis of
a,a-trehalose were recorded using a reported discontinu-
ous assay [17,43] where the formation of G1P was mea-
sured. The concentration of G1P was determined as
NADH produced in a second coupled enzymatic reaction
catalyzed by phosphoglucomutase and glucose 6-phos-
phate dehydrogenase. The reaction mixtures for phospho-
The phosphate site of Schizophyllum commune trehalose phosphorylase C. Goedl and B. Nidetzky
910 FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS
rolysis had a total volume of 200 lL and were incubated
in 1.5-mL tubes at 30 °C, using an Eppendorf Thermo-
mixer (Vienna, Austria) for temperature control and gen-
tle agitation using instrument settings of 300 r.p.m.
Typical enzyme concentrations used were 40 lgÆmL
)1
of
wild-type and 500 lgÆmL
)1
of R507A and K512A. The
reaction times varied between 0.1 h for wild-type enzyme
and up to 40 h for mutant phosphorylases. A plot of
concentration of G1P released against the incubation time
was linear in all cases, indicating that enzyme inactivation
did not interfere with determination of the initial rate
under the conditions used. Enzymatic rates (V) were
measured for conditions in which the concentration of
phosphate was varied in the range 5–300 mm whereas the
concentration of a,a-trehalose was 400 mm and constant.
Vanadate added in concentrations of 0.5, 2.5, or 5.0 mm

was tested as reversible inhibitor of phosphorolysis of
a,a-trehalose catalyzed by wild-type and mutant phos-
phorylases at pH 6.6. Inhibition constants (K
ic vanadate
)
were calculated using initial-rate data acquired under
conditions in which the concentration of phosphate was
varied at a constant concentration of a,a-trehalose
(400 mm) in the absence or presence of different constant
concentrations of vanadate.
Enzymatic rates of hydrolysis of a,a-trehalose or G1P
(V
hydrolysis
) were determined at 30 °Cin50mm Mes buffer,
pH 6.6, using 400 mm of disaccharide or 50 mm of sugar
1-phosphate substrate and measuring the concentration of
d-glucose released in samples taken at different times, up to
48 h. A hexokinase-based spectrophotometric assay was
used for the determination of d-glucose. Hydrolytic reac-
tions for wild-type phosphorylase were performed in the
absence and presence of 20 lm of vanadate.
Functional complementation studies for R507A
and K512A mutants
Initial rate assays in the direction of phosphorolysis of
a,a-trehalose were used to analyze restoration of activity
in K512A or R507A caused by the addition of an external
primary amine or a derivative of guanidine. Experiments
were carried out at 30 °Cin50mm Mes buffer, pH 6.6,
containing 50 mm of potassium–phosphate and 400 mm of
a,a-trehalose. The concentration of the amine or guanidine

derivative was typically 200 mM and constant, with the
exception of chemical rescue of R507A by guanidine and
K512A by propargylamine, which was analyzed at different
concentrations of external reagent in the range 10–200 mm.
Suitable controls showed that none of the added amines or
guanidines had a significant effect on the activity of the
wild-type enzyme incubated under otherwise exactly identi-
cal conditions to the wild-type enzyme alone. The increase
in ionic strength resulting from the addition of amine or
guanidine derivative was not corrected. However, the com-
parison of initial rates measured in the absence and presence
of NaCl in concentrations in the range 10–200 m m at
pH 6.6 and 7.5 clearly indicated that the activities of R507A
and K512A were not influenced by the relevant ionic
strength changes. The ratio k
rescue
⁄ k
0
, where k
0
and k
rescue
are V ⁄ [E] values determined in the absence and presence of
chemical rescue agent, respectively, is used to express the
degree of activation of the mutant. The pH dependence of
functional complementation of K512A by propargylamine
was determined in the pH range 6.6–8.5 using different
reagent concentrations in the range 10–200 mm.
Data processing
Processing of initial-rate data for the calculation of kinetic

parameters and inhibitor binding constants used reported
procedures [17]. Equation (1) was fitted to data from activ-
ity restoration experiments where k
max
is the maximum
initial rate, divided by [E], obtained at a saturating concen-
tration of the rescue agent, and K
R
is the half-saturation
constant for the reagent.
k
rescue
¼ k
max
Á½rescue agent=ðK
R
þ½rescue agentÞ þ k
0
ð1Þ
Acknowledgements
Financial support from the FWF Austrian Science
Fund (project DK Molecular Enzymology W901-B05)
is gratefully acknowledged. We thank Professor Walter
Keller (Department of Chemistry, University of Graz)
for help with CD spectroscopic analysis.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Conservation of active-site residues among
retaining glycosyltransferases of fold family GT-B dis-
played in a partial multiple sequence alignment.
Fig. S2. Comparison of CD spectra of wild-type (—),
R507A (- - -) and K512A (ÆÆÆÆÆ ) mutant trehalose phos-
phorylases.
This material is available as part of the online article
from .
Please note: Blackwell publishing are 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.
C. Goedl and B. Nidetzky The phosphate site of Schizophyllum commune trehalose phosphorylase
FEBS Journal 275 (2008) 903–913 ª 2008 The Authors Journal compilation ª 2008 FEBS 913