An insight into phosphorylase mechanism from model study

Article abstract of DOI:10.1080/15533174.2010.522550

Mechanistic aspects of phosphorylation reaction that mimic phosphorylase enzymes have been studied in the biologically important middle pH region by utilizing nitrophenol as substrate and bistrimethylenediaminecobalt(III) phosphate complexes as the enzyme model. Significant phosphorylation was noted from reactions of 1:1 molar ratio of nitrophenol and bistrimethylenediamincobalt(III) phosphate, [Co(III)tn₂Pi]. Enhanced phosphorylation was depicted for reaction solutions that contained 1:1 molar ratio of nitrophenol and di-bistrimethylenediamincobalt(III) phosphate, [(Co(III)tn₂)₂Pi]. Specific mechanistic features and the possible roles metal ions play in phosphorylase enzyme are highlighted. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15533174.2010.522550

Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 40:826–830, 2010
Copyright © Taylor & Francis Group, LLC
ISSN: 1553-3174 print / 1553-3182 online
DOI: 10.1080/15533174.2010.522550
An Insight into Phosphorylase Mechanism from Model Study
Fikru Tafesse and Kennedy Eguzozie
Department of Chemistry, University of South Africa, UNISA Pretoria, Republic of South Africa
pathways. Phosphorylases are enzymes that catalyze the addi-
Mechanistic aspects of phosphorylation reaction that mimic tion of a phosphate group from an inorganic phosphate to an
phosphorylase enzymes have been studied in the biologically im- acceptor. Note that a phosphatase removes a phosphate group
portant middle pH region by utilizing nitrophenol as substrate
and bistrimethylenediaminecobalt(III) phosphate complexes as the
enzyme model. Significant phosphorylation was noted from reac-
tions of 1:1 molar ratio of nitrophenol and bistrimethylenediamin-
cobalt(III) phosphate, [ Co(III)tn
2
Pi ]. Enhanced phosphorylation phosphate esters promoted by cobalt amine complexes, we
from a donor while a kinase transfers a phosphate group from a
donor (usually ATP) to an acceptor.
As a continuation of our research into the hydrolysis of
was depicted for reaction solutions that contained 1:1 molar ratio have reported the effect of metal ions and oxides of nitro-
of nitrophenol and di-bistrimethylenediamincobalt(III) phosphate,
gen in activating/inhibiting the reactions in model compounds.
[
2 2
(Co(III)tn ) Pi ]. Specific mechanistic features and the possible
4-Nitrophenyl-phosphate was utilized as the model substrate
roles metal ions play in phosphorylase enzyme are highlighted.
and bis-trimethylene- diaminecobalt (III) was employed as a
model for the metalloenzyme.[
4−7]
The possibility of phos-
Keywords cobalt(III) complexes, enzyme models, nitrophenol, ni-
phorylation occurring along with dephosphorylation was ini-
tially hypothesized in those reactions. In the present study,
our focus is geared to unraveling the phosphorylation reac-
tions that can be promoted in model systems that contain bis-
trimethylenediaminecobalt(III)phosphate and paranitrophenol
at neutral pH.
trophenylphosphate, phosphorylase, phosphorylation of
NP
1.
INTRODUCTION
Phosphate esters play a major role in the biological world by
linking processes of respiration and fermentation with other cel-
lular reactions. Enzyme catalyzed transfer of phosphoryl groups
is central to the energy balance for all organisms, and is also
involved in cellular control mechanisms of metabolism.[ The
aim of this study has been to establish those features of metal
ion complexation that are required for the promotion of phos-
phorylation reactions in metabolism. The achievement of this
goal is of some importance since the roles of the essential metal
ions in phosphorylase enzymes are not well understood. It has
already been established from model studies that the presence
of divalent metal ions is a requirement in hydrolysis reactions
4
- nitrophenol +Pi → 4 - nitrophenylphosphae
(
4−NP)
(4−NPP)
1]
The reactions are expected to mimic the actions of phos-
phorylase enzymes in biochemical processes. The results will
shed some light on the role of metal ions in the mechanism of
phosphorylase enzymes.
2
. MATERIALS AND METHODS
2
.1. Instruments and Reagents
All reagents used were either analytical reagent grade or the
purest available commercially and were used without further
purification. Measurement of pH was made with the Metrohm
[
2,3]
of phosphate esters.
Phosphorylation has a central role in
the regulation of numerous cellular processes. It is well estab-
lished that enzymes play a crucial role in those biochemical
632-pH meter equipped with a combination electrode.
A super aquarius 9000 series UV/vis spectrophotometer from
Cecil Instruments was used to obtain spectra collect rate data.
The pH of the reaction mixture was maintained by adding
Received 19 July 2010; accepted 21 July 2010.
National Research Foundation (NRF) is acknowledged for support-
ing research in our lab under ecotoxicology niche area. The assistance drops of 0.1M, 0.01M, and 0.001M of NaOH or HClO4
rendered to K. Eguzozie from Grow Your Own Timber Project (GYOT)
using different marked dotting glass rods in a simple tech-
of the College of Science engineering and technology, UNISA, is grate-
nique referred to as glass stick dotting. The diaqua com-
fully acknowledged.
3
+
plexes [Cotn2(H2O)2] where tn2 = bistrimethylenediamine
Address correspondence to Fikru Tafesse, Department of Chem-
istry, University of South Africa, P. O. Box 392 UNISA Pretoria, Re-
public of South Africa. E-mail: Tafesf@unisa.ac.za
were prepared in solution from the carbonato complexes
[Cotn2CO3] using published protocols.
+
[4,5]
The temperature
826

MODEL PHOSPHORYLASE MECHANISMS
827
of the reaction was controlled using a thermostated water bath, 10 ml in the thermostated reaction vessel. Similar protocols were
which allows the water to circulate via a jacketed reaction vessel. used to produce different concentrations of the metal phosphate
complexes.
2
.2. Protocol of the Study
2
.2.3. Reaction of Nitrophenol with Preformed Cobalt(III)bis-
trimethylenediamine Phosphate
Two mL of an aqueous solution of nitrophenol (NP) was
2.2.1. Reaction of Nitrophenol with Phosphate
The protocol for the study of the reaction of 1:1 molar ratio
of nitrophenol (NP) and inorganic phosphate (Pi) consists of
mixing 2 mL of an aqueous solution of nitrophenol (NP) and
2
mixed with two mL of an aqueous solution of the preformed
cobalt (III) phosphate complex in the thermostated reaction ves-
sel at 25 C. The concentration of the two reactant solutions was
identical. The pH was maintained at 7 for the whole duration
of the reaction (30 minutes) under constant stirring. After 30
minutes of reaction, aliquots were withdrawn for the recording
of uv/vis spectra.
mL of an aqueous solution of sodium dihydrogen phosphate
◦
(Pi) with the required concentration, in the thermostated reaction
◦
vessel at 25 C. Six mL of deionized water was added to it and
the pH maintained at 7 for the whole duration of the reaction
under constant stirring. After 30 minutes of reactions Aliquots
were withdrawn for the recording of uv/vis spectra. For different
nitrophenol to inorganic phosphate ratio (NP: Pi), the amount
of NP is held constant (2 ML) and that of Pi varied in the total
reaction solution of 10 mL.
3
. RESULTS
3.1. Calibration Curve Studies of Nitrophenol and
Nitrophenylphosphate
2.2.2. Preparation of Cobalt (III) Bis Trimethylenediamine
Phosphate Complexes
Calibration curve studies were done by preparing a series
(
Cotn2Pi,(Cotn2)2Pi,(Cotn2)3Pi). (Please note that charges of 4-nitrophenol and 4-nitrophenyl phosphate solutions rang-
−3
−6
and degree of protonation is omitted in formulae of all of the ing from a concentration of 10 to 10 M, and the pH ad-
−3
complexes for clarity). A solution that contains 1 × 10
M
justed to 7.0. The spectra are depicted in Figures 1 and 2. Under
cobalt (III) phosphate is prepared by mixing two mL of an aque- the pH of study, the 4-nitrophenol is deprotonated to form the
−3
ous solution of 5 × 10 M cobalt(III) bis trimethylenediamine 4-nitrophenolate ion, which is bright yellow and has an ab-
−3
diaqua complex with the same amount of 5 × 10 M sodium sorbance maximum at 400 nm, quite distinct from NPP, which
dihydrogen phosphate (Pi) in the thermostated reaction vessel has an absorbance maximum (λ max) at 310nm. In a system
◦
at 25 C. Six mL of deionized water was added to it and the pH where the pH is maintained, the ratio of the concentration of
maintained at 7 for the whole duration of the reaction (30 min- the p-nitrophenolate ion to the p-nitrophenol would be constant
utes) under constant stirring. Similarly the preparation of 1 × allowing for Beers law to operate.
−3
10
M solutions of (Cotn2)2Pi and (Cotn2)3Pi was undertaken
The absorbance values at 310 and 400 nm were plotted
by mixing 4 and 6 ml of the cobalt(III)bis trimethylenediamine against concentration and the molar absorptivity (ε) determined.
diaqua solution, respectively, to make up the total volume to The molar absoptivity (ε) for 4-nitrophenol at 310 and 400 nm
FIG. 1. Absorption spectra of nitrophenol solutions at pH 7.0.

828
F. TAFESSE AND K. EGUZOZIE
FIG. 2. Absorption spectra of nitrophenylphosphate solutions at pH 7.0.
was calculated from the above plot as 6398.9 LMol 1cm−1 and absorbance values at 310 nm for the various aliquots over the
8
−
−1
−1
876.3 LMol cm respectively. Similarly the molar absorp- absorbance value of the same concentration of nitrophenylphos-
tivity value for nitrophenylphosphate at 310 nm was found to be phate at the same wavelength. Alternatively, the absorbance val-
1098 Lmol cm . Triplicate analysis was done for each re- ues at 400 nm for the various aliquots over the absorbance value
−1 −1
1
action. The standard deviation for the triplicate analysis ranged of the same concentration of nitrophenol may be used to deter-
from 0.1 to 0.2. The average value was used for analysis of the mine the percentage phosphorylation. Note that in the first case
data.
it is an increase in the amount of nitrophenylphosphate that is
being monitored, while in the latter case it is the depletion of ni-
3.2. Phosphorylation Studies
trophenol that is measured. The calculation afforded comparable
The results of phosphorylation studies are depicted in Table 1. values in both scenarios. The different cobalt –phosphate com-
The percentage phosphorylation was calculated from the net plexes do not affect the uv spectra of the measured nitrophenol
TABLE 1
◦
Percentage phosphorylation for the reactions of [(Cotn2)xPi] (where x = 1,2,3. charges omitted) with NP at 25 C and pH 7.0
Final concentration of
each reactant [ ] f
% phosphorylation
calculated at 400 nm
% phosphorylation
calculated at 310 nm
Reactants
−
−
−
−
3
3
4
4
NP + Cotn2Pi
2 × 10
33.5
32.1
25.6
22.2
17.2
15.1
70.0
68.4
60.3
56.8
40.1
34.6
32.4
25.3
22.4
20.4
14.5
12.9
36.4
35.7
25.6
22.2
17.4
15.2
72.7
71.4
60.7
56.9
40.1
34.9
37.3
29.8
22.5
20.5
14.8
12.9
1
× 10
× 10
× 10
2
1
2
1
−
5
.5 × 10
−
5
5
5
.25 × 10
−
3
NP + (Cotn2)2Pi
NP + (Cotn2)3Pi
2 × 10
−
3
1
2
1
2
× 10
× 10
× 10
−4
4
−
−
5
.5 × 10
−
1
.25 × 10
−
3
2 × 10
−
3
1
2
1
2
1
× 10
× 10
× 10
−4
4
−
−
5
.5 × 10
.25 × 10−

MODEL PHOSPHORYLASE MECHANISMS
829
imental condition showed a decrease in the absorbance maxima
at 400 nm and an increase at 310 nm. These changes are consis-
tent with the assertion that NP is being depleted and NPP being
formed in the solution. An increase of pH during the forma-
−
tion of the Co(III)tn2Pi is attributed to the release of OH from
the monodentate complex to produce the four member chelate.
Formation of the cobalt(III)Pi complex results in a decrease of
electron density on the phosphorous center increasing its elec-
trophilic character. Hence, the attack of the phosphorous center
by oxygen of the nitrophenolate ion (NP) is enhanced to pro-
duce NPP. Similarly the addition of 2:1 and 3:1 molar ratio of
Co(III)tn2(OH)(H2O) and Pi under the experimental condition is
presumed to produce largely (Co(III)tn2)2Pi and (Co(III)tn2)3Pi,
respectively, as evidenced previously.[ In these complexes, the
additional metal ions per phosphate will force the phosphorous
center to adapt an increased electrophilic character, thereby fa-
cilitating the attack of NP on phosphorous. The results show an
increased percentage phosphorylation for NP + (Co(III)tn2)2Pi.
8]
SCH. 1. Proposed reaction scheme for the reaction of nitrophenylphosphate
with bis trimethylenediaminecobalt(III) phosphate (charges and degrees of pro-
tonation omitted for clarity).
and nitrophenylphosphate as they are not uv active in the wave- A decrease in the percentage phosphorylation for the reactions
length of interest at millimolar concentrations.
of NP + (Co(III)tn ) Pi is a result of both hydrolysis and phos-
2
3
phorylation reactions to be occurring simultaneously in the re-
action solutions as the reaction conditions favor hydrolysis than
condensation reaction. The above reactions mimic the activa-
4. DISCUSSION
In our experiments it was observed that freshly pre- tion and deactivation of phosphorylase enzymes by metal ions
pared solutions containing cis-hydroxoaqua bistrimethylenedi- in biological systems.
aminecobalt(III) ions exhibit different visible spectral character-
istics from those that were prepared and kept in the refrigerator
5
.
CONCLUSION
In the current study, the role of divalent metal ions in phos-
for several days. Enhanceddifferences in thevisiblespectrawere
noted at increased concentrations. This observation is consistent
with dimerization of the cis- hydroxoaquaions by olation reac-
tions that result in the formation of singly and doubly bridged µ-
hydroxo products. Addition of Co(III)tn2(aq) at pH 7.0, where
Co(III)tn2(OH)(H2O) predominates to phosphate at 1:1molar
ratio results in rapid water substitution to form the monodentate
complex which can then form a four membered chelate. The
latter has been shown to be more stable than the ring opened
phorylation reactions has been highlighted. Some of the difficul-
ties experienced in previous in situ investigations such as those
limited to labile metal ions, have been circumvented by mak-
ing use of partially inert Co(III) complexes with well defined
coordination geometry. Specific mechanistic features revealed
−
in this study include the requirement of cis coordinated OH
on the metal center for high reactivity and the full chelation of
the phosphate oxygens by the metal complex for effective at-
tack of the substrate ( nitrophenolate ion) on the metal complex
to result in phosphorylation. This observation has similarity
with that documented for biochemical processes.[ Synthe-
sis and breakdown of glycogen occur by different paths, cat-
alyzed by different enzymes. For synthesis, the cell uses an
activated intermediate, UDP-glucose. Conversely, breakdown
produces glucose 1-phosphate. Glycogen is broken down princi-
pally by glycogen phosphorylase. This enzyme (usually known
simply as "phosphorylase") uses the cofactor pyridoxal 5’-
phosphate (PLP) to cleave glycogen phosphorolytically. Note
that the reaction is not hydrolytic as no water is used in
the cleavage reaction. Instead, inorganic phosphate combines
with the nonreducing terminal glucose residue to give glucose
[
8,9,10]
monodentate complex at pH 7.
However, complete quan-
titative formation of the 1:1 chelates, even in relatively concen-
trated solutions, may not be achievable because of: (1) olation of
hydroxoaqua ions, which decreases the effective concentration
of the monomeric hydroxoaquaspecies; and (2) formation of
higher metal to phosphate complexes which accompany the for-
mation of the 1:1 complex. Similarly, addition of Co(III)tn2(aq)
at pH 7.0 to phosphate at 2:1 and 3:1molar ratio results in the
formation of several chelates of which the predominant species
are (Cotn2)2Pi and (Cotn2)3Pi, respectively. A mixture of mon-
odentate coordinated and four member ring chelate species are
anticipated in the reaction solution. The presence of the dif-
11]
31
ferent species in the solution has been detected by P NMR
studies.[8]
1-phosphate.
The reaction of nitrophenolate (NP) with phosphate(Pi)
showed no remarkable change in the absorption characteris-
tics of the the uv/vis spectra. The addition of NP to a preformed
Glycogen + Pi −→ Glucose-1-phosphate + Glycogen
(n-residue)
1
:1 molar ratio of Co(III)tn2(OH)(H2O) and Pi under the exper-
(n-1 residue)

830
F. TAFESSE AND K. EGUZOZIE
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that lead to the synthesis and breakdown of the above reac-
tions is of a paramount importance. Our study has highlighted
the role of metal complexes in the phosphorylation reactions
of biochemical processes that mimic phosphorylase enzymes.
Scheme 1 depicts the overall reactivity as summed up below:
The reaction of inorganic phosphate (Pi) with bis trimethylene-
diaminecobalt(III) produces bistrimethylene- diaminephospha-
tocobalt(III) in solution. The phosphorous center thus becomes
more electrophilic compared to uncoordinated Pi. The attack
of the metal coordinated phosphorous center by the oxygen of
nitrophenol (NP) results in phosphorylation to produce nitro-
phenylphosphate (NPP). Note that degree of protonation and
charges of the complexes are omitted for clarity in the above
scheme.
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Abbreviations
tn
Pi
NP
NPP
= trimethylenediamine NH2CH2CH2CH2NH2
= inorganic phosphate
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of hydroxide on coordinated 4-nitrophenyl phosphate. J. Amer. Chem. Soc.
= nitrophenol
1
983, 105, 7327–7336.
= nitrophenylphosphate
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3+
2+
Co(III)(aq) = [Cotn2(H2O)2] or [Cotn2(OH)(H2O)]
ed.; Freeman, W. H., 2010, pp 603–605.

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