Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose kcatP¼ 0.033 s1 and substrate binding affinity in the ternary complex with e
Trang 1phosphorylase through the characterization of wild-type and His334fiGly mutant enzymes
Alexandra Schwarz1, Lothar Brecker2 and Bernd Nidetzky1
1 Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria
2 Institute of Organic Chemistry, University of Vienna, Austria
Glycogen phosphorylases are pyridoxal 5¢-phosphate
(PLP)-dependent glycosyltransferases (EC 2.4.1.1) that
catalyze the reversible phosphorolysis of oligomeric
and polymeric a-1,4-glucan substrates (maltodextrins,
starch, glycogen) [1,2] The reaction proceeds with retention of configuration at the anomeric carbon, yielding a-d-glucose 1-phosphate (Glc1P) as product
in the direction of substrate depolymerization In spite
Keywords
a-retaining glucosyl transfer; phosphorus
NMR; pyridoxal 5¢-phosphate; saturation
transfer difference NMR; starch
phosphorylase
Correspondence
B Nidetzky, Institute of Biotechnology and
Biochemical Engineering, Graz University of
Technology, Petersgasse 12, A-8010 Graz,
Austria
Fax: +43 316 873 8434
Tel: +43 316 873 8400
E-mail: bernd.nidetzky@tugraz.at
(Received 15 May 2007, revised 1 August
2007, accepted 6 August 2007)
doi:10.1111/j.1742-4658.2007.06030.x
His334 facilitates catalysis by Corynebacterium callunae starch phosphory-lase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate We have substituted His334 by a Gly and measured the disrup-tive effects of the site-directed replacement on acdisrup-tive site function using steady-state kinetics and NMR spectroscopic characterization of the cofac-tor pyridoxal 5¢-phosphate and binding of carbohydrate ligands Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose (kcatP¼ 0.033 s)1) and substrate binding affinity in the ternary complex with enzyme bound to phosphate (Km¼
280 mm), respectively The 31P chemical shift of pyridoxal 5¢-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate
At pH 7.25, it was not sensitive to the replacement His334fi Gly Analysis
of interactions of a-d-glucose 1-phosphate and a-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from satura-tion transfer difference NMR experiments, suggested that disrupsatura-tion of enzyme–substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6 pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5–7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5–7.0) External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction It caused displacement of the entire pH profile of H334G by + 0.5 pH units
A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced
Abbreviations
CcStP, Corynebacterium callunae starch phosphorylase; GL, D -gluconic acid 1,5-lactone; Glc1P, a- D -glucose 1-phosphate; LFER, linear free energy relationship; PLP, pyridoxal 5¢-phosphate; STD, saturation transfer difference; X1P, a- D -xylose 1-phosphate.
Trang 2of detailed studies spanning many decades, definite
conclusions about the catalytic mechanism of glycogen
phosphorylases and the exact function of the PLP
co-factor in it are still elusive [2–6] Figure 1 shows that
an active site His has a central role in the contentious
debate surrounding a putative covalent glucosyl–
enzyme intermediate of a double displacement-like
mechanism of the phosphorylase The precedent of
sucrose phosphorylase [7–9], mechanistically
represent-ing a large class of retainrepresent-ing glycoside hydrolases and
transglycosidases, would strongly favor some form of
a two-step mechanism, consisting of glucosylation and
deglucosylation of a catalytic group on the enzyme,
typically a carboxylate of Glu or Asp [5,10] Glycogen
phosphorylase structures reveal that the backbone
amide carbonyl of the His is the only group
appropri-ately placed to function as a nucleophile [4,11–15]
(Fig 1B) However, despite the vast assortment of
probes used, all searches for a covalent intermediate of
glycogen phosphorylase have proved fruitless so far
[5] Partly driven by this negative evidence, an
alterna-tive mechanism termed SNi-like was proposed, where,
in the direction of phosphorolysis, attack of phosphate
as nucleophile and departure of the oligosaccharide
leaving group occur on the same face of the glucosyl
residue being transferred [2,4–6,11,13] It involves only
a single transition state that has a highly developed
oxocarbenium ion character The His is proposed to
stabilize this transition state through electrostatic and
hydrogen bonding interactions of its main chain
car-bonyl and side chain, respectively The phosphate ion
positioned in a ‘tucked-under’ conformation on the
opposite (a) face of the glucosyl oxoarbenium ion-like
species presumably provides additional electrostatic stabilization, derived from its interactions with C-1 and O-5 as well as O-2 of the pyranosyl ring [4,6,11,13] (Fig 1B) Earlier kinetic studies of wild-type phosphorylases support this idea by showing coopera-tive-like (synergistic) binding of phosphate and gluco-syl oxoarbenium ion mimics such as d-gluconic acid 1,5-lactone (GL) [16,17] Substitution of His334 in starch phosphorylase from Corynebacterium callunae (CcStP) (Fig 1A) by Gln or Asn caused a substantial (up to 150-fold) loss in wild-type catalytic efficiency that was paralleled by a corresponding decrease in affinity for GL in combination with phosphate, reflect-ing a change from positive to negative cooperativity in binding of the two ligands as a result of the site-direc-ted replacement [18]
In this work, we have substituted His334 with Gly and analyzed the disruptive effects of the point muta-tion on active site funcmuta-tion of CcStP using steady-state kinetics and selective NMR probes for the 5¢-phos-phate group of the cofactor and for bound carbo-hydrate ligands The work was carried out to address three questions in particular, taking into account that, quite unexpectedly, an H334A mutant of CcStP was almost as active as the wild-type enzyme [18] How does complete removal of the side chain of His334 influence binding and catalysis? Are the properties of neighboring active site groups, including the PLP cofactor, affected by the Hisfi Gly mutation? If suffi-cient room is vacated in H334G to accommodate water or another nucleophile in place of the original methylimidazole group, will this new ligand participate
in the enzymatic reaction such that eventually
hydro-N
O N
N
O Pyridoxal P O O OH
O
O O
OH OH
O P O O O
N O
N O N
O
O N
N
O
O O N
O
O N
O
N O O
His345 (334)
Gly114 (114)
Leu115 (115) Gly640 (629)
Glu637 (626)
Tyr538 (527)
Asn449 (437)
Ser639 (628)
3.0 Å
3.1 Å
2.9 Å 2.9 Å
3.6 Å 3.6 Å
3.6 Å 2.7 Å
3.1 Å 3.7Å
2.7 Å
2.8 Å 3.2 Å
4.3 Å 3.2 Å
B A
Fig 1 Close-up structure of the active site of CcStP and proposed interactions with Glc1P bound at the catalytic subsite (A) The picture was generated with PYMOL v.0.99 using X-ray crystallographic coordinates for CcStP with phosphate bound in the active site (Protein Data Bank entry 2C4M) His334, PLP and phosphate are shown as stick models (B) The scheme was drawn using the structure of E coli malto-dextrin phosphorylase bound to Glc1P (Protein Data Bank entry 1L5V) Numbering of amino acids is for the E coli enzyme, and correspond-ing residues of CcStP are given in parentheses Hydrogen bonds are indicated as broken lines.
Trang 3lysis or transglucosylation occurs? Mutational analysis
of the His homologous to His334 in CcStP has not
been performed in another a-glucan phosphorylase
Results
Protein purification and cofactor analysis
Wild-type CcStP and the H334G mutant were
pro-duced in Escherichia coli and purified to apparent
homogeneity (data not shown) Both enzymes were
obtained in similar yields of about 50%, and contained
approximately 0.8 PLPs per subunit of protein Upon
excitation at 330 nm, the wild-type enzyme and the
H334G mutant exhibited nearly superimposable
cofac-tor fluorescence emission spectra between 470 and
550 nm, with an emission maximum at 520 nm
How-ever, the intensity of cofactor fluorescence at 520 nm
in the H334G mutant was only approximately 40%
that observed in the wild-type
Characterization of the H334G mutant
Enzyme activity
The H334G mutant exhibited 0.003% of the wild-type
specific activity for phosphorolysis of maltodextrin
(33 UÆmg)1) External imidazole stimulated activity of
the mutant up to 5.5-fold, whereas it weakly inhibited
the wild-type (Fig 2) Acetate and formate had no
effect on the activity of the H334G mutant Azide,
2-methylimidazole and 2-ethylimidazole inhibited the
mutant The wild-type was inhibited weakly (£ 2-fold)
by all of the compounds tested, with the exception of
formate, which caused a five-fold reduction of activity
Kinetic parameters Steady-state kinetic parameters for phosphorolysis of maltopentaose by the H334G mutant were determined
at pH 7.0 under conditions where the concentration of phosphate was constant and saturating (50 mm) The
kcat of 0.033 ± 0.001 s)1 was 0.05% of the wild-type value The Km for maltopentaose was 280 ± 20 mm, reflecting a 75-fold decrease in substrate binding affin-ity as a result of the mutation Like the wild-type [18], the H334G mutant did not hydrolyze maltopentaose into glucose above a detection limit of about 0.15% of its phosphorylase activity
Ligand binding Dissociation constants (Kd) for complexes of the H334G mutant with GL or Glc1P were obtained from nonlinear fits of a Langmuir binding isotherm to data obtained by fluorescence titration analysis The Kd values were 95 ± 8 lm and 100 ± 10 lm for complexes with Glc1P and GL, respectively They were decreased seven-fold and three-fold in comparison to Kd values for corresponding complexes of the wild-type [18] The presence of 50 mm phosphate promoted a 30-fold increase in Kd(¼ 2.9 ± 0.3 mm) for GL binding to the H334G mutant This result is in contrast to the 17-fold enhancement of GL binding to the wild-type upon the addition of the same concentration of phosphate
pH profiles The pH dependences of logarithmic rates of the H334G mutant and wild-type are compared in Fig 3 Data for the wild-type are taken from Griessler et al [19] The pH profile of the H334G mutant in the phos-phorolysis direction was a narrow, bell-shaped curve, strikingly different from that of the wild-type and with
an optimum pH of 6.5 A shift of the pH profile of about + 0.5–1.0 pH units and an optimum pH similar
to that of the wild-type was observed for the H334G mutant in the presence of 200 mm imidazole By con-trast, the pH rate profile of the wild-type was not affected by addition of the same concentration of imid-azole (data not shown)
Phosphorus NMR of pyridoxal 5¢-phosphate
31P-NMR spectra for solutions of wild-type CcStP and the H334G mutant that contained a similar concentra-tion of enzyme-bound PLP ( 100 lm) were recorded
in the pH range 5.6–8.0 Typical spectra acquired at
pH 7.25 are shown in Fig 4A The 31P resonance
imidazole (mM) 0
0
2
4
6
Fig 2 Analysis of restoration of activity in wild-type CcStP (d) and
the H334G mutant (s) by external imidazole The results are given
as relative specific activities that were normalized by using the
spe-cific activities of the wild-type (33 UÆmg)1) and the H334G mutant
(0.001 UÆmg)1) in the absence of imidazole.
Trang 4signal of PLP phosphate in the H334G mutant showed
a very low signal-to-noise ratio, necessitating data
col-lection for up to 12 h, during which time a perceptible
denaturation of the enzyme occurred at pH values
below and above 7.25 It was therefore not possible to
obtain an exact pH dependence for the 31P chemical
shift of PLP phosphate in the H334G mutant
How-ever, a single31P shift at pH 7.25 is provided
Figure 4B compares pH profiles of chemical 31P
shifts for PLP phosphate in wild-type CcStP measured
in the absence and presence of 20 mm sodium arsenate
The two pH profiles were almost superimposable on
each other We also determined chemical 31P shifts at
pH 6.68 and 6.93 under conditions in which the
pres-ence of arsenate (20 mm) and GL (1 mm) drives
formation of a ternary enzyme–ligand complex The
results show that 31P shifts for PLP phosphate in free enzyme were remarkably insensitive to the binding of arsenate alone and in combination with GL
Analysis of ligand binding by STD NMR Figure 5 summarizes relative saturation transfer differ-ence (STD) effects of Glc1P and a-d-xylose 1-phos-phate (X1P) upon their binding to wild-type and H334G phosphorylase Glc1P displayed very similar patterns of binding to both enzymes However, the relative STD effects of the protons in positions 6a and b were slightly higher when Glc1P was bound to the wild-type than when it was bound to the H334G mutant The relative STD effects of X1P bound to the two enzymes were also fairly similar, with the exception of the proton in position 5eq, which showed
a higher effect in the complex with the H334G mutant Binding of GL to the wild-type and the H334G mutant also yielded very similar STD spectra with, however, quite a low signal-to-noise ratio, very likely caused by the small dissociation constants for enzyme–GL complexes Appreciable STD effects could
be detected only for protons in positions 2 and 4, which caused overlapping signals in the 1H-NMR spectrum (data not shown) [20] All other protons showed much lower STD effects, which could not be quantified Although longer STD measurements could,
in principle, improve the signal-to-noise ratio, the duration of the NMR experiment was limited in this case by the spontaneous hydrolysis of GL to gluconic acid During STD NMR measurements of Glc1P bound to wild-type enzyme, we observed formation of
a novel carbohydrate at the expense of Glc1P This compound was analyzed directly from the NMR sample, and identified as amylose (data not shown) Details underlying the conversion of Glc1P in the
pH
31P (p.p.m.)
1 2 3 4
Fig 4 Characterization of PLP phosphate in wild-type CcStP and the H334G mutant using 31 P-NMR (A) Spectra of wild-type CcStP and the H334G mutant acquired at
pH 7.25, and with the number of recorded scans and resulting signal-to-noise ratios indicated (B) Chemical31P shifts of the PLP phosphate resonance signal of wild-type enzyme in the absence of ligand (d), in the presence of 20 m M arsenate (s), and in the presence of 20 m M arsenate and 1 m M GL (.); 31 P shift for the H334G mutant (,), recorded in the absence of arsenate and at only a single pH of 7.25.
pH 5.5
kcat
1.4
1.6
1.8
2.0
Fig 3 pH profiles of catalytic rates for phosphorolysis of
maltodex-trin catalyzed by wild-type CcStP (.) and the H334G mutant in the
absence (d) and presence (s) of 200 m M imidazole The initial
rates were acquired under conditions of apparent saturation with
substrate, and are given as relative values (rel kcat) of the catalytic
rate for the wild-type (50 s)1; pH 7.0) and the catalytic rates of the
H334G mutant in the absence (0.0015 s)1; pH 6.5) and the
pres-ence (0.0081 s)1; pH 7.0) of imidazole The lines indicate the trend
of the data.
Trang 5absence of an exogenous glucosyl acceptor
oligosac-charide were not pursued further
Discussion
Disruptive effects of active site mutations traced
by STD NMR
Interpretation of the functional consequences of
H334G and active site mutations of enzymes in general
is subject to the caveat that site-directed replacement
has caused a global change in enzyme–substrate
inter-actions occurring in the wild-type There is a clear
need for practical methods capable of characterizing
the structural perturbation resulting from site-specific
modification of enzyme or substrate with respect to
direct as well as indirect disruptive effects caused by it
We would like to suggest the STD NMR technique,
which analyzes, in the dissociated ligand, the
magneti-zation transferred from protons of the protein to
pro-tons of the bound ligand that are in close contact with
the protein Relative STD effects within a given ligand
therefore provide a characteristic fingerprint of
nonpo-lar ligand interactions within the binding pocket of the
protein [21–27] Because hydrogen bonds and other
electrostatic interactions are silent in the STD NMR
experiment, the obtained portrait of the binding
pat-tern is partial (Fig 1B), and isolated interpretations of
STD effects can therefore be hazardous However, if
STD effects for two minimally modified systems can
be investigated and compared, then the interpretation
is considerably simplified The side chain of His334
and the –CH2OH group of Glc1P are complementary
interacting groups (Fig 1B), and analysis of changes
in relative STD effects resulting from structural pertur-bation of enzyme (H334G) and substrate (X1P) was therefore of particular interest The results obtained suggest an overwhelmingly local disruption of binding interactions caused by removing the two functional groups individually or together
Analysis of kinetic consequences in the H334G mutant and chemical rescue studies
Substitution of His334 with Gly caused a 103.5-fold decrease in the wild-type kcat for phosphorolysis of maltopentaose Conversion of the ternary enzyme–sub-strate complex is believed to be the rate-determining step of glucosyl transfer to phosphate catalyzed by a-glucan phosphorylases [1], and kcatP is the kinetic measure of it Because substrate binding to enzyme– phosphate is supposed to be a rapid equilibrium pro-cess [1,28], the Km for maltopentaose is an effective dissociation constant that was increased by almost two orders of magnitude in the H334G mutant in relation
to the wild-type Comparison of different CcStP mutants reported here (H334G) and in a recent paper (H334A, H334Q, H334N [18]) reveals that complete removal of the His side chain in the H334G mutant had the largest disruptive effect on both binding and turnover of maltopentaose Unlike the H334A mutant,
in which the kinetic consequences of the site-directed replacement were minimal [18], the H334G mutant had lost 30 kJÆmol)1 of the binding energy used in the wild-type for stabilization of the transition state of the reaction (The differential binding energy DDG# was
Fig 5 Analysis of sugar 1-phosphate
bind-ing to wild-type CcStP and the H334G
mutant using STD NMR Values are relative
STD effects of Glc1P bound to wild-type
CcStP (Aa) and the H334G mutant (Ab) as
well as X1P bound to wild-type CcStP (Ba)
and the H334G mutant (Bb) Each STD
effect is calculated as a quotient of signal
intensities in the STD spectrum and in the
reference proton spectrum The effects are
normalized to the respective largest effect
in the sample.
Trang 6calculated with the relationship DDG#¼ RT ln 105.2,
using the ratio of kcatP⁄ Km values of 18 000 m)1Æs)1
and 0.12 m)1Æs)1 for the wild-type and the H334G
mutant, respectively.) We speculated that water might
occupy the position vacated in the H334A mutant
through removal of the imidazole group of the His,
thereby effectively replacing the function of the
origi-nal side chain in catalysis by the mutant [18]
What-ever mechanism truly accounts for the retention of
phosphorylase activity by the H334A mutant, it is
clearly not available to the H334G mutant The
selec-tivity of the H334G mutant for glucosyl transfer to
phosphate as compared with water was absolute within
the limits of detection of the experimental methods,
suggesting that, as in the wild-type and the H334A
mutant [18], water was effectively excluded from the
reaction with maltopentaose bound to free enzyme or
enzyme–phosphate
The notion that substitution of His334 with Gly
destabilizes the transition state of glucosyl transfer but
otherwise does not alter the course of the reaction
cat-alyzed by CcStP is further supported by the results of
linear free energy relationship (LFER) analysis and
chemical rescue studies Schwarz et al [18] have shown
that a log–log correlation of catalytic efficiencies of the
wild-type and His334 mutants for phosphorolysis of
starch with the corresponding reciprocal dissociation
constants for GL binding to enzyme–phosphate was
linear, with a good coefficient of determination Using
a similar type of correlation, which is now based on
kcatP⁄ Km for maltopentaose and includes data for the
H334G mutant, we obtain again a plausible LFER
with a slope of 1.93 ± 0.45 and a coefficient of
deter-mination (r2) of 0.862 (supplementary Fig S1) A shift
in the controlling mechanism of the reaction brought
about by the Hisfi Gly mutation would be expected
to cause a breakdown of the LFER, in contrast to the
observations made Whereas external imidazole weakly
enhanced the activity of the H334G mutant, it did not
participate in the reaction as alternative nucleophile,
such that glucose 1-imidazole or the product of its
spontaneous hydrolysis (glucose) would be formed in
kinetic competition with Glc1P Other small
nucleo-philes, such as azide, were without effect on both
activity and reaction course By way of comparison,
when the catalytic nucleophile (Asp) of sucrose
phos-phorylase was replaced by Ala, azide could occupy the
position of the original carboxylate group and react
through addition to C-1 of the glucosyl moiety,
yield-ing the inversion product b-glucose 1-azide [9]
We investigated whether the proposed hydrogen
bond between His334 and the C-6 hydroxy group of
the glucosyl residue bound at the catalytic subsite
could become optimized in the transition state A hypothetical scenario, inspired by studies of human purine nucleoside phosphorylase [29,30], is that His334 could be responsible for positioning O-6 in line with O-5 and the glycosidic oxygen of phosphate (OP1) (Fig 6) In the direction of polysaccharide synthesis, compression of the three-oxygen stack such that O-6 moves closer to the ring oxygen would enhance elec-tron density in the reactive carbon and thus facilitate glycosidic bond cleavage and formation of the transi-tion state in an SNi-like mechanism of glucosyl trans-fer In the direction of phosphorolysis, both O-6 and the now nucleophilic OP1 of phosphate might be pushed towards O-5 and assist electronically in cataly-sis As in purine nucleoside phosphorylase [29,30], protein vibrations that are coupled to the reaction coordinate could be responsible for promoting the close approach of the three oxygens
pH rate dependences for the wild-type and the H334G mutant examined with kinetics and
31P-NMR
As for other a-glucan phosphorylases [31–35], the pH profiles of apparent kcat for wild-type CcStP were
ND-1 2.7 Å
2.3 Å
3.0 Å
3.8 Å
O-6
O-5
O P1
Fig 6 Suggested role for the hydrogen bond between Nd of His334 and the 6-OH of the glucosyl residue bound at the catalytic subsite in the selective stabilization of the transition state O-6, the ring oxygen, and the glycosidic oxygen OP1lie in a close three-oxygen stack that is indicated by a dashed line Increased electron density near the reactive center provided by squeezing the three oxygens together could facilitate the catalytic step The picture was generated using Protein Data Bank entry 1L5V (maltodextrin phos-phorylase bound with Glc1P [15]).
Trang 7bell-shaped curves showing a decrease in activity at
low and high pH Replacement of His334 with Gly
caused a marked change in the pH profile of kcat for
the phosphorolysis direction To explore possible
sources of the different pH dependences, we used
31P-NMR and compared chemical shifts for the
5¢-phosphate group of PLP in the wild-type and the
H334G mutant Changes in chemical shift and line
width of the 31P-NMR signal may serve as reporters
of alterations in the ionization state of the cofactor
phosphate group [36] They are, however, also
expli-cable by changes in the local environment of PLP
and their effect on conformational strain on the
5¢-phosphate moiety
The 31P chemical shift of PLP phosphate in
unli-ganded wild-type CcStP was strongly influenced by pH,
increasing in a sigmoidal dependence from 1.36 p.p.m
at pH 5.6 to 3.66 p.p.m at pH 8.0 (Fig 4B) Slow
deprotonation of the triethanolamine buffer interfered
with measurement of31P chemical shifts in the alkaline
region (pH > 7.5), preventing determination of a
complete pH profile for the chemical shift and hence
calculation of the pKavalue of PLP phosphate by curve
fitting However, there is good evidence that the pKa
value for PLP phosphate in free CcStP is ‡ 6.75
(Fig 4B), and therefore higher than that seen in
maltodextrin phosphorylase (pKa¼ 5.6) [37,38]
To the extent that the shift of the 31P resonance
signal is a sensitive probe of direct contacts between
the cofactor 5¢-phosphate group and bound ligands
or relevant changes in active site conformation
induced by ligand binding [2,38], the evidence for
CcStP suggests that the local environment of PLP
phosphate remains essentially unaffected upon
forma-tion of enzyme complexes with arsenate alone and in
combination with GL By contrast, significant field
shifts of the 31P resonance signal were observed with
E coli maltodextrin phosphorylase [37], potato
phos-phorylase [39] and muscle glycogen phosphos-phorylase [40] upon addition of arsenate, probably caused by electrostatic interactions between the 5¢-phosphate moiety and arsenate The pKa for PLP phosphate in
E coli maltodextrin phosphorylase was also shifted
by + 1.1 pH units upon binding of arsenate [37] Therefore, CcStP appears to differ subtly from mal-todextrin and glycogen phosphorylase in how it copes with constraining the cofactor phosphate group into a configuration that is believed to promote catalysis via direct interaction with the substrate arsenate (or phosphate) A tentative explanation is provided by Fig 7, which reveals clear differences in the pattern
of hydrogen bonding and the orientation of PLP phosphate in the active sites of CcStP bound with phosphate (Fig 7A) and maltodextrin phosphorylase bound with phosphate and a nonphosphorolyzable substrate analog (omitted in Fig 7B for reasons of clarity) in Fig 7B Gly642 in the E coli enzyme is substituted by Ser631 in CcStP Interactions from the main chain amide of Gly are replaced by interactions from both the main chain amide and the side chain
of Ser Hydrogen bonds between PLP phosphate and the side chains of nearby Lys residues and bound phosphate ligand appear to be stronger in maltodex-trin phosphorylase than in CcStP, arguably account-ing for the relative elevation of pKa of PLP phosphate in unliganded CcStP and the apparent lack
of perturbation of pKa in the CcStP complex with arsenate
The pH dependence of the31P chemical shift of PLP phosphate in wild-type CcStP is not, clearly, borne out
in pH rate profiles for the enzymatic reaction The opti-mum pH range for glucosyl transfer to and from phos-phate overlaps with the pH region (pH 6.0–7.0) where monoanionic and dianionic forms of 5¢-phosphate should both be present in similar relative amounts The loss of wild-type activity in the direction of synthesis at
Fig 7 Comparison of the sites for PLP
phosphate in CcStP (A) and E coli
malto-dextrin phosphorylase (B) Pictures were
generated using Protein Data Bank entries
2C4M (CcStP) and 1L5W (maltodextrin
phosphorylase bound with phosphate and a
substrate analog [15]).
Trang 8pH 6.5 [19] may be correlated, at least formally, with
the strong field shift of31P resonance signal in this pH
range, perhaps reflecting the formation of a PLP
dian-ion Electrostatic repulsion may now prevent the
cofac-tor 5¢-phosphate and also the dianionic phosphate of
the glucosyl donor substrate from closely approaching
each other [2,41]
Rather than eliminating a single ionization from
pH profiles, substitution of His334 by Gly caused a
complex pattern of changes in the pH rate
dependenc-es of the wild-type The acidic and basic limbs on the
pH profile of the H334G mutant for the
phosphoroly-sis direction were displaced inward by 0.5 pH units
in comparison with the corresponding pH profile of
the wild-type, and the optimum pH range for the
mutant was also shifted, by about ) 0.75 pH units In
addition to partly restoring activity in the H334G
mutant, external imidazole caused an upshift by £ 1.0
pH units of the entire pH dependence of
phosphoro-lysis by the mutant, whereas the pH rate profile of
the wild-type was not influenced by the added
imidaz-ole Although these results suggest that His334
influ-ences the pH dependence of the activity of CcStP,
they do not delineate a detailed relationship
Appar-ent ionizations on the pH rate profiles must probably
be assigned to pH-dependent ‘titration’ of more than
just a single residue
Experimental procedures
Materials
Materials for mutagenesis, protein purification and
enzy-matic assays have been described elsewhere in more detail
[18,42] Restriction endonucleases were obtained from
Fer-mentas (St Leon-Rot, Germany) Oligonucleotide synthesis
and DNA sequencing was performed at VBC Biotech
Ser-vices GmbH (Vienna, Austria) All other chemicals were of
the highest quality and were provided by Sigma-Aldrich
(Vienna, Austria)
Mutagenesis, protein expression and purification
PCR-based overlap extension method [43] PCR conditions
were as described previously [42], except for an annealing
and elongation time of 1 min and an annealing temperature
mutagenic primers, where Eco91I and XagI restriction sites
are underlined and the mismatched codons are indicated
in bold, respectively: XagI-for, 5¢-GGGAACTCTGCGCCT
GTTACCCAATC-3¢; H334G-for, 5¢-TACACCAACGGAA
CCGTGCTCAC-3¢; H334G-inv, 5¢-GTGAGCACGGTTC CGTTGGTGTACGC-3¢
The plasmid pQE70–CcStP [42] containing the gene for wild-type CcStP was used as the template The mutagenized plasmid was transformed into E coli JM109 cells Protein expression and purification of the H334G mutant were car-ried out using published protocols [18] Enzyme activity was measured with a continuous coupled assay reported elsewhere [9], and protein was determined by the Bio-Rad (Vienna, Austria) dye binding assay using BSA as standard
Steady-state kinetic analysis and biochemical characterization
Initial rates of phosphorolysis were determined in discontin-uous assays as described previously [44] The enzyme (5.5 lm
300 mm potassium phosphate buffer, and the release of Glc1P was measured as a function of time of incubation up
to 3 h Maltodextrin or maltopentaose was used as the sub-strate, as indicated in Results The sodium salts of azide, ace-tate, and formate, as well as imidazole, 2-ethylimidazole, and 2-methylimidazole, were tested in the range 10–250 mm for possible restoration of activity of the H334G mutant for
Con-trol reactions with the wild-type were carried out in all cases The H334G mutant was examined for possible hydrolase
50 mm triethanolamine buffer (pH 7.0), containing malto-pentaose (75 mm) and potassium sulfate (20 mm) Note that sulfate was added in this series of measurements to ensure stability of the enzymes during the timespan of experiments carried out in the absence of phosphate [45] Samples were taken at certain time points up to 40 h, and the formation of glucose was measured as described elsewhere [18]
pH dependence studies were performed in the pH range 5.5–8.0 The pH values were adjusted at the temperature of
enzymatic reaction Ionic strength changes in the pH range examined were not considered Catalytic rates of the H334G mutant were acquired under conditions of apparent
mal-todextrin)
Apparent dissociation constants for the binary complex
of the H334G mutant bound with Glc1P or GL, and for the ternary complex of the H334G mutant bound with phosphate and GL, were determined by titration analysis in which quenching of the PLP fluorescence was measured Following excitation at 330 nm, an emission spectrum in the range 350–550 nm was recorded The full experimental protocol and details of data processing for the calculation
of dissociation constants are given elsewhere [18] The PLP content of isolated H334G mutant was measured with a quantitative spectrophotometric test [46]
Trang 9NMR spectroscopy
DRX 600 AVANCE spectrometer using topspin 1.3
soft-ware (Bruker) Proton, carbon and phosphorus spectra
242.94 MHz, respectively The one-dimensional spectra
were recorded with 32 768 data points Zero filling to
65 536 data points, appropriate exponential multiplication
and Fourier transformation led to spectra with ranges of
P)
employ-ing a slight modification of a reported procedure [37]
Samples were prepared by adding 150 lL of 400–450 lm
enzyme solution in 50 mm triethanolamine buffer,
contain-ing 20 mm potassium sulfate, to 450 lL of a solution
con-taining 50 mm triethanolamine buffer, 50 mm acetate and
used, sulfate does not inhibit the enzyme activity through
competition with phosphate, suggesting that occupancy of
measured with proton decoupling and 2048 scans and a
preacquisition delay of 1.0 s, resulting in spectra with a
sig-nal-to-noise ratio of about 10 : 1 to 20 : 1 after about 1 h
Two-dimensional spectra were obtained from 256
experi-ments, each with 2048 data points and an appropriate
num-ber of scans Zero filling and Fourier transformation led to
proton and carbon, respectively Chemical shifts have
STD NMR spectra were measured employing a reported
procedure [27] Samples were prepared in 5 mm potassium
well as 5 mm ligand Before addition to the NMR tube, the
enzyme storage solution was gel-filtered using NAP 5
col-umns (GE Healthcare, Vienna, Austria) equilibrated with
hundred and twelve scans were collected, each with 50
Gaussian-shaped pulses (50 ms and 1 ms delay) and a
spectral width On and off resonance irradiations were
subtraction was performed via phase cycling, and no water
suppression was applied Reference proton spectra were
recorded with 256 scans directly before and after the STD
measurements
Acknowledgements
Financial support from the Austrian Science Fund
(FWF P15208-B09, P18038-B09 and P15118) is
grate-fully acknowledged
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