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Bioorganic Chemistry 66 (2016) 4145

Contents lists available at ScienceDirect

Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

NMR studies on mechanism of isomerisation of fructose 6-phosphate to


glucose 6-phosphate catalysed by phosphoglucose isomerase from
Thermococcus kodakarensis
Shahzada Nadeem Abbas a,d, Kenneth Hun Mok b, Naeem Rashid a, Yongjing Xie b, Manuel Ruether c,
John OBrien c, Muhammad Akhtar a,
a

School of Biological Sciences, University of the Punjab, Lahore, Pakistan


School of Biochemistry and Immunology, Trinity College, University of Dublin, Ireland
c
School of Chemistry, Trinity College, University of Dublin, Ireland
d
Department of Biology, Lahore Garrison University, DHA, Lahore, Pakistan
b

a r t i c l e

i n f o

Article history:
Received 28 September 2015
Revised 2 March 2016
Accepted 13 March 2016
Available online 14 March 2016
Keywords:
Phosphoglucose isomerase
Thermococcus kodakarensis
Hyperthermophilic
Nuclear magnetic resonance
Mechanism
Isomerization

a b s t r a c t
The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P)
was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis
(TkPGI) through 1D and 2D NMR methods. When the reaction was performed in 2H2O the hydrogen
atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization
occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to
those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.
2016 Elsevier Inc. All rights reserved.

1. Introduction
The aldose-ketose interconversion of sugar phosphates, 1  3
(Scheme 1), is carried out by isomerases. Historically glucose
6-phosphate and triose phosphate isomerase (Scheme 1) from
rabbit muscle (eukaryotes) have been subjected to extensive
mechanistic and stereochemical studies [15]. With both these
enzymes the reaction has been established to occur by a
cis-enediol intermediate as shown in 2, Scheme 1. Key features of
such a mechanism are the loss of one of the two pro-chiral hydrogen atoms from the methylene group of the ketose sugar and the
incorporation of a medium derived hydrogen atom at C-2
(Scheme 2) of aldose, as exemplified by the reaction catalyzed by
triose phosphate isomerase [1,4].
The loss into and incorporation from the medium of a hydrogen
atom,1 in such conversions, is based on the assumption that the conjugate acid formed in the deprotonation step, exchanges its proton
Corresponding author.
E-mail address: ma3@soton.ac.uk (M. Akhtar).
Hydrogen refers to all the isotopes of this element, while, protium, deuterium and
tritium are specific designations.
1

http://dx.doi.org/10.1016/j.bioorg.2016.03.004
0045-2068/ 2016 Elsevier Inc. All rights reserved.

with the medium hydrogen atoms faster (7  9) than it is transferred to the enediol intermediate (7  8), thus it is the species 9
that forms the product (10).
Balance between the intramolecular and exchange-mediated
pathways of Scheme 3 is governed by several factors, most significant of which is the temperature, as highlighted by the rabbit
muscle isomerase catalysed conversion of [1-3H] F6P into G6P.
Here, at 0 C, 83% of the reaction occurs by the intramolecular
pathway while at 60 C about 75% of the conversion takes the
exchange-mediated course [6]. While in the case of the reaction
catalyzed by triose phosphate isomerase, 98% of the reaction takes
the exchange-mediated course [4]. In the latter reaction, it was
estimated that the exchange 7  9 is 50-times faster than the protonation step 78.
In contrast to the aforementioned two isomerases, work on
D-xylulose-D-xylose isomerase showed that this rearrangement
predominantly involves an intramolecular hydrogen transfer2
between C-1 and C-2 of the pentose skeleton, Scheme 4 [7,8]. The
assumption thus was that with this enzyme the intramolecular

2
Hydrogen transfer is a general term and does not specify whether this atom is
transferred as proton, hydride or a radical.

42

S.N. Abbas et al. / Bioorganic Chemistry 66 (2016) 4145

H
H

OH

OH

isomerisation was rationalized to involve an intramolecular hydride


transfer [10]. In the light of this precedent, it began to be felt that
there are two classes of isomerases, metal-free and metalcontaining; the former operating by an enediol mechanism and the
latter by a hydride transfer. The influence of this generalization
was so strong that when the X-ray structure of phosphoglucose isomerase from Pyrococcus furiosus (PfPGI) was determined, the authors
hypothesized a hydride transfer mechanism for the enzyme, because
of the presence of a metal ion near O1/O2, ignoring the fact that Glu97
was located near the hydrogen atoms bound to C-1/C-2, which could
act as a base in the enediol mechanism [11]. Subsequently, the
enzyme was studied by Berrisford et al. [12], who showed that the
isomerization does not occur by an intramolecular hydrogen transfer
but by an enediol process (of the type 6  7  9, Scheme 3), in
which Glu97 acts as a base [12]. We have recently cloned a metaldependent phosphoglucose isomerase, from a hyperthermophilic
organism, Thermococcus kodakarensis, which is optimally active at
90 C and requires Zn2+ for its activity [13]. The aim of this study
was to evaluate whether a metal dependent enzyme, that is T.
kodakarensis phosphoglucose isomerase, operates by a hydride shift
or an enediol process. In this paper, we have studied this problem
using 1D and 2D NMR.

OH

H
O

OH

HO

O
2C

OH

OH

Scheme 1. Enzyme catalyzed reversible inter-conversion of aldoses into ketoses.


R = a, is the C-3 substituent of glyceraldehyde 3-phosphate and R = b the C-3 to C-6
skeleton of glucose 6-phosphate respectively.

pathway is favored over the exchange-mediated alternative because


the architecture of the active site during catalysis remains shielded
from the surrounding medium, thus forbidding the exchange reaction of the type 7  9 (Scheme 3). Then, from X-ray crystallographic
studies on D-xylose isomerase it was deduced that while at the active
site of the enzyme, a metal ion was available, in the vicinity of the O1
and O2, to act as an electrophile for the activation of the carbonyl
group, no satisfactory basic group could be seen around the C-1,
C-2 hydrogen atoms to act as base for proton removal in the mechanism involving an enediol intermediate [9]. Consequently, the

2. Materials and methods


2.1. Materials
F6P, G6P as sodium salts and rabbit phosphoglucose isomerase
(200 U/mg) were purchased from Sigma. Recombinant phosphoglucose isomerase from T. kodakarensis (TkPGI) was produced by
molecular cloning and gene expression by the methods already
described [13].

Hpro-S

OH
Haq+

Hpro-R

OH

Haq

O
R

Scheme 2. Exchange-mediated inter-conversion catalyzed by triose phosphate isomerase, showing the incorporation of a medium hydrogen (H+aq) at C-2.

OH

B:

A
H

:A

Intramolecular path

B:

O
H

An exchange path
with H2O

OH

A
H

Haq

Haq

B:
H

:A

OH

Haq

A
H

10

Scheme 3. Postulated mechanism of aldose-ketose isomerases. In the exchange-mediated path the hydrogen abstracted by the enzymic base is exchanged with the protons
of the medium and then transferred to the intermediate 9.

43

S.N. Abbas et al. / Bioorganic Chemistry 66 (2016) 4145


o

Hpro-S

OH

Hpro-R

O
HO

OH

HO

OH

then dissolved in the same volume of 2H2O to make deuterated


phosphate buffer). 1D and 2D 1H: - 13C HSQC (hetero nuclear single
quantum coherence) spectra of the aforementioned reaction mixtures were analysed on Bruker NMR Avance using operating frequency of 600 MHz at 25 C.

OH

OH

3. Results and discussion

OH

D-xylulose

The 1D-proton NMR spectrum of G6P in 2H2O in Fig. 1 (trace a)


shows that the anomeric hydrogen atoms of the a- and b-anomers
are located at 5.17 and 4.6 ppm respectively, as duplets [12]. The
C-2 hydrogen is coupled on the one hand to the C-1 hydrogen atom
and on the other with that at C-3 and appears as a multiplet, while
the C-3 hydrogen at 3.66 ppm is also a multiplet. Addition of T.
kodakarensis isomerase to the latter followed by incubation at
90 C for 2 h, resulted in a dramatic change of these resonances;
now these regions contain only singlets for the two anomeric protons and show complete disappearance of the C-2 proton (trace b,
Fig. 1). Furthermore, the resonance for the C-3 hydrogen also
shows a change. The shifts may be attributed to the exchange of
the C-2 proton of G6P with deuterons of the medium. In addition,
control experiments run in the absence of the isomerase showed
that, over the same time period, no incorporation of deuterium into
any of the two anomers occurred (trace a, and c, Fig. 1).
Analysis of the F6P resonances by 1D NMR (data not shown),
when the sample was treated with T. kodakarensis isomerase in
2
H2O, at 90 C for 2 h, showed that the peaks corresponding to its
H-1pro-R and H-1pro-S overlapped, to a large extent, with signals
from the hydrogens of the isomerized G6P. Therefore, in case of

D-xylose

11

12

Scheme 4. The intramolecular hydrogen transfer in the reaction catalyzed by


D-xylulose-D-xylose isomerase.

2.2. Methods
10 mg each of F6P and G6P was, separately, incubated for 2 h
with 1 mg of TkPGI at 90 C in 50 mM deuterated phosphate buffer,
pH 6.5, in an overall reaction volume of 600 lL. In parallel experiments, 10 mg each of F6P and G6P was, incubated for 1/2 h with
50 U (60.25 mg) of rabbit PGI at 25 C in 50 mM deuterated
phosphate buffer, pH 7, in an overall reaction volume of 600 lL
(Phosphate buffer of the required pH was prepared in simple water
(1H2O) using the pH meter calibrated with H2O based buffers. Later
the buffer sample was vacuum dried to evaporate all the water and

0.07
0.06

H-1a

H-2b

4.59

0.04

H-3a

H-1b

Doublet
to
Singlet

0.03
3.21

0.02

3.66

5.17

Normalized Intensity

0.05

(a)

0.01
0
-0.01

(b)

-0.02

(c)

-0.03
5.0

4.5

4.0

3.5

Chemical Shift (ppm)

O
O
6

O
O

HO3

O H
4

OH

O
O OH

H
OH

OH HO 3

Alpha D-Glucose 6- phosphate

H
1

H
2

O
6

H
OH

H
4

H
2

OH

Beta D-Glucose 6- phosphate

Fig. 1. 600 MHz 1D NMR spectra of (a) G6P in 2H2O; (b) the latter treated with TkPGI and (c) F6P in 2H2O without enzyme. All the samples were treated at 90 C for 2 h. The
spectra highlight the regions containing the two anomeric C-1 hydrogens and those at C-2 and C-3 of G6P. The other details are in the experimental section and the formulae
above show the structures of a- and b-anomer.

44

S.N. Abbas et al. / Bioorganic Chemistry 66 (2016) 4145

F1 Chemical Shift (ppm)

(a)
60

0.05

62

0.26

64

66
3.60

3.55

3.50

3.45

3.40

3.45

3.40

F2 Chemical Shift (ppm)

F1 Chemical Shift (ppm)

(b)
60
0.09

0.02

62

64

66
3.60

3.55

3.50

F2 Chemical Shift (ppm)

F1 Chemical Shift (ppm)

(c)
60

62

64
Pro-S

H-1
66

Pro-R

Pro-R

H-1

-F6P
3.60

Pro-S

H-1

H-1
-F6P

3.55

3.50

3.45

F2 Chemical Shift (ppm)


Fig. 2. 600 MHz 2D 13C1H HSQC spectra of (a) F6P and rabbit PGI in 50 mM phosphate buffer, pH 7, in 2H2O at room temperature, (b) F6P and TkPGI in 50 mM phosphate
buffer, pH 6.5, in 2H2O at 90 C and (c) untreated F6P in 50 mM phosphate buffer, pH 6.5 in 2H2O at 90 C. The numbers above the resonance profile in traces (a) and (b) show
integrals. The other details are in the experimental section.

F6P, deuterium incorporation was determined using 2D NMR;


13
C1H heteronuclear single quantum coherence (HSQC). The two
diastereotopic hydrogen atoms the a- and b-anomer of F6P gave
distinct chemical shifts at 3.573.63 and 3.463.53 ppm respectively (Fig. 2, trace c); the corresponding published values being
3.623.68 and 3.533.58 ppm [12]. The 13C frequencies were at
64.2 and 62.8 ppm for the a- and b-anomer, compared to 65.6
and 65.3 ppm in Ref. [12]. By and large the values obtained in
the present study are similar to those reported in the literature,
though there is greater divergence for the 13C chemical shifts for
which we offer no explanation, except that in Ref. [12] a more
powerful 800 MHz instrument was used. In the presence of the T.
kodakarensis isomerase in 2H2O, these are replaced by a single resonance for each anomer of F6P (Fig. 2 trace b), showing that one of
the C-1 hydrogen atoms of F6P must have been replaced by deuterium. In order to explore the stereochemistry of the hydrogen
replaced, advantage was taken of the known stereochemistry of
the reaction catalysed by rabbit muscle phosphoglucose isomerase.
The comparison of the profile in Fig. 2, trace with b, shows that the
anomeric hydrogens had similar but not identical chemical shifts,
which may be attributed to the use of different pH buffers for
the two enzymes necessitated by their specific pH optima.

N:

His

158

N:

His

2+

Zn
H

O
Glu

16

H
O

O
H

158

2+

Zn
H

97

O
Glu

Zwitterion state

97

17

Scheme 5. Architecture of the active-site as proposed in Ref. [14], leading to the


formation of the Zwitterion, 17.

Notwithstanding this minor anomaly, the nearly identical shift


for the C-1 anomeric hydrogen atoms suggests that rabbit muscle

S.N. Abbas et al. / Bioorganic Chemistry 66 (2016) 4145

and T. kodakarensis isomerases have identical steric course. Since it


is pro-R hydrogen at C-1 of F6P that is exchanged by the rabbit
muscle enzyme [5] the same course is followed by T. kodakarensis
isomerase. The exchange of the C-2 hydrogen atom of G6P (Fig. 1)
and C-1 pro-R hydrogen of F6P (Fig. 2) with protons of the medium
by T. kodakarensis isomerase is consistent with the requirements of
an enediol mechanism, operating via an exchange-mediated path,
involving the sequence of the type 6  7  9  10, Scheme 3.
Similar results were previously obtained with phosphoglucose
isomerase of P. furiosus [12]. In the latter case, Glu97 was identified
as the base that abstracts the proton. The P. furiosus enzyme has
82.4% similarity with the isomerase from T. kodakarensis as shown
by the amino acid alignment in Table 1S. which shows that the
equivalent residue in T. kodakarensis isomerase is also Glu97. By
analogy with the study in Ref. [12] the base involved in the
abstraction of the proton by T. kodakarensis isomerase, is also
deduced to be Glu97.
T. kodakarensis isomerase as well as that catalyzing the interconversion of D-xylulose and D-xylose (Scheme 4) are archaeal
metalloproteins, which highlights the fact that the presence of
metal electrophile to activate the carbonyl group is required not
only for the hydride shift mechanism but also for the operation
of an enediol process.
P. furiosus isomerase has also been subjected to molecular
dynamics simulations and it is proposed that Zn2+ is involved in
a structural, and not a catalytic, role (Scheme 5) [14]. The paper
[14] departs from the earlier view [11,12], on the architecture of
the enzyme active-site and suggests that Glu97 points towards
the carbonyl oxygen while X-ray data have been interpreted to
place it near the C1/C2 hydrogen bonds [11,12]. Notwithstanding
this debatable suggestion, an appealing aspect of the proposal
[14] is that a common carbocation (17) has been invoked to be
involved in the enediol as well as hydride shift mechanisms. Such
a proposal brings the two processes as minor variations on a common theme in the sense that a carbocation of the type 17 has the
propensity either to be quenched by a hydride transfer or a proton
removal. The environmental factors that determine the choice
between the two options are not known at present.
4. Conclusions
The status of hydrogen atoms at C-2 of G6P and C-1 of F6P was
studied in the reaction catalysed by TkPGI. The isomerization
occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. The results have suggested that the presence of metal
electrophile to activate the carbonyl group is required not only
for the hydride shift mechanism but also for the operation of an
enediol process.
Conflict of interest
The authors declare no conflict of interest.

45

Acknowledgment
We are thankful to Higher Education Commission of Pakistan
for providing funds to S.N. Abbas under International Research
Support Initiative Program.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.03.
004.
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