Isotope selective activation: A new insight into the catalytic activity of urease

Article abstract of DOI:10.1039/c7ra05489k

Urease, a metalloenzyme, requires carbon dioxide (CO₂) for its activation. But, whether this activation is isotope-specific to ¹²CO₂ or ¹³CO₂, is not yet known and even the potential role of CO₂ in the enzymatic activity of urease is poorly understood. Here, we provide direct experimental evidence that the catalytic activity of urease exhibits a unique isotope-specific response where the ¹²CO₂ isotope is strongly preferred over the ¹³CO₂ isotope during its catalytic activation. Moreover, this isotope-selective activation depends on different isotopic fractionations (¹²C:¹³C) of the reaction-environment as well as the substrate urea (¹³C-urea and ¹²C-urea), where the ¹²CO₂ isotope in the reaction medium essentially facilitates the hydrolysis of ¹³C-enriched urea. This deepens our understanding of the isotope-specific urease activation and its potential role in hydrolytic reaction. Our findings thus may offer novel opportunities for a better fundamental understanding of isotope-specificity in chemical reactions involving metalloenzymes.

Full text of DOI:10.1039/c7ra05489k

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Isotope selective activation: a new insight into the
catalytic activity of urease
Cite this: RSC Adv., 2017, 7, 31372
Sanchi Maithani,† Mithun Pal,† Abhijit Maity and Manik Pradhan *ab
a
a
a
Urease, a metalloenzyme, requires carbon dioxide (CO
2
) for its activation. But, whether this activation is
, is not yet known and even the potential role of CO in the
enzymatic activity of urease is poorly understood. Here, we provide direct experimental evidence that
12
13
isotope-specific to CO
2
or CO
2
2
12
the catalytic activity of urease exhibits a unique isotope-specific response where the CO
2
isotope is
13
strongly preferred over the CO
2
isotope during its catalytic activation. Moreover, this isotope-selective
12
13
activation depends on different isotopic fractionations ( C: C) of the reaction-environment as well as
13
12
12
2
the substrate urea ( C-urea and C-urea), where the CO isotope in the reaction medium essentially
Received 15th May 2017
Accepted 14th June 2017
13
facilitates the hydrolysis of C-enriched urea. This deepens our understanding of the isotope-specific
urease activation and its potential role in hydrolytic reaction. Our findings thus may offer novel
opportunities for a better fundamental understanding of isotope-specificity in chemical reactions
involving metalloenzymes.
DOI: 10.1039/c7ra05489k
rsc.li/rsc-advances
8
9
10
reaction kinetics, action of buffers and effect of temperature.
But the mechanism underlying the activation of urease or its
Introduction
Urease, the rst enzyme to be isolated in crystalline form, was potential role in hydrolytic reaction still remains controversial.
1,2
found to have a dinuclear nickel(II) active site. This Ni(II)- Moreover, it was demonstrated in the past that the metal-
11,12
containing metalloenzyme is found in various soil microbes, loenzyme urease requires CO
2
for its activation.
Therefore,
bacteria, fungi and plants. It plays an important role in envi- the product of the urease-catalyzed hydrolysis of urea and the
3
ronmental nitrogen transformations. Urease is also a potent activator of the catalyst (i.e. CO
2
) being the same entity makes
virulence factor in certain pathogens and is particularly known the reaction mechanism more complex. In this context, the
1
3
for its potential link to human diseases like peptic ulcers and detailed study of isotopic signatures of CO
(i.e. CO
) would provide better insight into the reaction mecha-
] and nisms. However, so far there have been no studies focused on
subsequently has found widespread applications in enzymatic the potential role of isotope-specic CO environment on the
assays and as urea sensors for routine clinical measurements of isotopic-fractionations of in situ CO production in the urease–
urea in blood, urine and different body uids. Besides this, it urea reaction. Moreover, how the variation of isotopic compo-
and
2
2
1
2
3,4
stomach cancer. Moreover, this enzyme acts as a unique
catalyst for its very specic action on urea [CO(NH
CO
2
2 2
)
2
2
5,6
1
2
13
13
12
5
is also widely used in waste water treatment for removal of urea
and as a model enzyme for the study of enzymatic kinetics.
sitions ( C: C) of the substrate urea ( C-urea and C-urea)
affects the catalytic activity of urease particularly in the envi-
5
The urease-catalyzed hydrolysis of urea has extensively been ronment of atmospheric CO
studied in the past. Urease catalyzes the hydrolysis of urea to explored and therefore remains an open question. Conse-
concentration, has never been
2
5,7
produce ammonium carbamate [H
2
NCO
2
NH
4
] which rapidly quently, unravelling the reaction mechanism involving the
À
decomposes into bicarbonate [HCO
3
] and ammonium ions isotope-selective activation of urease would open new perspec-
for urease
+
[
NH ] in a non-enzymatic and buffer-mediated system. The tives for a better understanding of the role of CO
2
4
bicarbonate nally gets converted into carbon dioxide (CO2). activation kinetics.
Early studies focused on several aspects of this reaction such as
In this study, we report for the rst time, that the enzymatic
activity of urease exhibits a unique isotope-selective CO2
affinity. Furthermore, we have showed that the catalytic activity
of urease depends on the isotopic compositions of both
reaction-environment and substrate. Finally, we also demon-
strate that the isotope-specic activation of urease is buffer-
a
Department of Chemical, Biological and Macromolecular Sciences, S. N. Bose
National Centre for Basic Sciences, Salt Lake, JD Block, Sector III, Kolkata-700098,
India. E-mail: manik.pradhan@bose.res.in; manik.pradhan@gmail.com; Fax: +91
3
3 2335 3477; Tel: +91 33 2335 5706; +91 33 2335 5708
b
Technical Research Centre (TRC), S. N. Bose National Centre for Basic Sciences, Salt independent. This new knowledge is of great signicance for
Lake, JD Block, Sector III, Kolkata-700098, India
fundamental understanding of isotope-specic chemical reac-
tions involving metalloenzymes. Fig. 1 illustrates a scheme
†
Both authors contributed equally to this work and should be considered as joint
rst authors.

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1
2
Fig. 1 A scheme showing the isotope-specific catalytic reaction of urease enzyme. (a) Isotopic CO
2
concentration deceases with simultaneous
13
13
12
in situ generation of CO
2
isotope in the urease-catalyzed hydrolysis of C-urea, (b) urease specifically requires CO
2
, exhibiting no intake of
13
13
2 2
CO even in an enriched CO reaction-environment.
1
3
13
2
showing the isotope-specicity of urease enzyme and specic generation of CO in the urease-catalyzed hydrolysis of C-
1
2
13
requirement of CO
2
isotope for its activation.
urea. We also interestingly (Fig. 2a) observed that while C-
urea derived in situ CO
with increasing initial headspace concentration of CO
same time the nal headspace CO
1
3
2
production is increased gradually
1
2
2
, at the
2
concentration is decreased
Result and discussion
12
more in the reaction. Taken together, these observations indi-
cate that there is a potential link between the enzymatic activity
To investigate the urease (1 mM)-catalyzed hydrolysis of isotope
enriched C and C-urea (4 mM) at different reaction envi-
ronments, we rst altered the headspace of the reaction with
1
2
13
of urease and the requirement of specic isotope of CO (i.e.
2
1
2
1
2
2
CO ). These observations are indistinguishable for urease-
increasing concentration of CO
2
(with ꢀ1.1% natural abun-
-free pure N environment. It
is noteworthy to mention here that only the concentration of
1
2
1
3
catalyzed hydrolysis of C-urea because the product and the
dance of CO
2
) starting from CO
2
2
1
2
headspace component are the same isotopic species (i.e. CO
Another striking nding revealed in our observations
Fig. 2b) is that in CO -free pure N environment, the in situ
generation of CO concentration from C-urea was observed
2
).
1
2
13
CO or CO isotopes is regulated keeping the total pressure
2
2
(
2
2
constant at around atmospheric pressure above the reaction
1
3
13
1
2
2
medium. We observed (Fig. 2a) that the headspace CO2
1
2
to be the lowest value, whereas the C-urea derived in situ
concentration was absorbed with simultaneous in situ
1
3
13
12
12
Fig. 2 Urease (1 mM)-catalysed hydrolysis of (4 mM) C-urea ($99% C) and C-urea ($99% C) at different headspace concentrations (partial
12
13
12
13
13
pressures) of CO
2
. (a) CO
has been increased whereas C-urea derived CO
dotted region shows the CO -free pure N headspace condition.
2 2
is generated while headspace CO concentration has been absorbed in the hydrolysis of C-urea. (b) C-Urea
13
12
12
12
derived CO
2
2 2
has been diminished with increasing headspace CO concentration. The
2
2
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1
3
13
12
12
Fig. 3 Urease (1 mM)-catalysed hydrolysis of (4 mM) C-urea ($99% C) and C-urea ($99% C) at enriched headspace concentrations (partial
13
12
13
13
12
pressures) of CO
2
(
CO
2
:
CO
2
: 40% : 60%). (a and b) Depict the variation of CO
urease-catalysed hydrolysis of C-urea and C-urea, respectively.
2
and CO
2
concentrations before and after the reaction of
1
3
12
1
2
12
production of CO
2
concentration was found to be markedly even for urease (1 mM)-catalyzed hydrolysis of (4 mM) C-urea.
1
2
enhanced. However, in absence of any CO
activity of urease diminishes and subsequently the hydrolysis of links to CO for its activation, thus unveiling a missing link
C-urea becomes very small, resulting in lower amount of between the enzymatic activity of urease and the necessity for
CO production. In contrast, C-urea derived in situ CO2
2
, the catalytic Our observation clearly manifests that urease preferentially
1
2
2
1
3
1
3
12
12
12
CO isotope.
2
2
itself facilitates the enzymatic activity of urease and thus urease-
Next, we examined the catalytic activity of urease in response
1
2
12 13
2
catalyzed hydrolysis of C-urea is not hindered in CO -free pure to the different isotopic-compositions ( C: C) of the substrate
1
2
2
N environment. However, C-urea derived in situ production urea to gain a better insight into the fundamental processes of
1
2
of CO
tration of CO
partial pressure of the same species CO above the reaction isotopic-compositions ( C: C) were achieved by suitably mix-
2
decreases with increase of initial headspace concen- isotope – selective nature of urease with a xed headspace
1
2
2
and this is likely to be the combined effect of atmospheric CO
2
concentration (ꢀ360 ppm). The different
1
2
12 13
2
1
2
13
medium as well as the over-saturation of urease enzyme in ing C-urea and C-urea, while retaining the same concen-
1
2
presence of excess headspace CO2.
tration of urea (4 mM). We observed (Fig. 4a) that the in situ
1
3
We next altered the headspace environment with ꢀ60% (103 generation of the product CO
2
from hydrolysis of 1 : 2
), (instead of ( C : C) isotopic-composition of urea was almost identical to
1
3
12
13
ppm) isotope enriched CO
2
(of 174 ppm total CO
2
1
2
13
13
ꢀ
1.1% natural abundance as in the previous case), to ensure the that of 1 : 99 ( C : C) isotopic-composition. However, C-
1
2
12
13
specic role of CO
Fig. 3a) that initial headspace CO
absorbed at all, while the headspace CO2 was markedly ( C : C) isotopic-compositions. This observation demon-
2
in the catalytic activity of urease. We found isotopic enrichment of the substrate urea for 1 : 2 ( C : C)
1
3
(
2
concentration was not isotopic-composition was signicantly lower than that of 1 : 99
1
2
12
13
1
2
12
decreased in the usual way for urease (1 mM)-catalyzed hydro- strates that the in situ availability of CO derived from the C-
2
1
3
13
lysis of (4 mM) C-urea. Moreover, under this altered environ- urea, itself activates urease for higher hydrolysis of C-fraction
1
2
13
mental conditions in headspace, no noticeable effect of of the 1 : 2 ( C : C) isotopic-composition of urea, whereas the
1
3
2
enriched headspace CO concentration was observed (Fig. 3b)
12
13
Fig. 4 Urease (1 mM)-catalysed hydrolysis of (4 mM) different isotopic compositions ( C: C) of substrate urea at a fixed head-space
1
3
12 13
concentration of CO
2
(ꢀ360 ppm). (a) Demonstrates the CO
2
evolved in the hydrolysis of (4 mM) different isotopic compositions ( C: C) of
13
13
urea. (b) Depicts a comparison between % content of C isotope in urea and CO
2
produced in the reaction with urease.
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12
13
Fig. 5 Concentration dependent study of urease-catalysed hydrolysis of C-urea and C-urea. (a and b) Elucidate the effect of different
concentrations of urea and urease, respectively.
1
3
12
13
12
13
C-urea ( C : C: 1 : 99) was not effectively hydrolysed in concentration (ꢀ360 ppm). The product ( CO
2
or CO
2
) of (1 mM)
in the reaction medium. urease-catalyzed hydrolysis of C-urea and C-urea was found
In order to gain a detailed and in-depth understanding of the (Fig. 5a) to be gradually increased with increasing concentration
1
2
12
13
absence of in situ generation of CO
2
1
2
12
13
2
role of CO derived from different isotopic fractionations of of urea ( C-urea or C-urea) from 100 mM to 5 mM. The obser-
1
2
13
C-urea, we then normalized the in situ generation of CO2 vation was followed by a sharp decline aer 5 mM urea concen-
from the different isotopic-compositions of urea (i.e. 2 : 1, 1 : 1 tration, thus exhibiting the effect of substrate-inhibition on the
1
3
and 1 : 2) to the percentage (%) of CO
2
produced from 99% urease-catalyzed hydrolysis. In contrast, keeping the urea
1
3
C-urea (i.e. 1 : 99) and this is illustrated in Fig. 4b. It was concentration xed (4 mM), there was no signicant change of the
1
3
12
13
2 2 2
found (Fig. 4b) that the percentage of in situ CO production concentration of the product ( CO or CO ) with increasing
was much higher than the C percentage actually present in all concentration of urease enzyme (Fig. 5b), thus indicating that
1
3
the different isotopic-compositions of urea, suggesting the urease still hydrolyzes urea in an efficient way and thereby indi-
1
3
inadequate hydrolysis of C-urea (1 : 99) in absence of suffi- cates the steady catalytic activity of urease. However, it is note-
1
2
cient in situ CO2 concentration in the reaction medium. worthy that the isotope-specic activation of urease was observed
Therefore, urease-catalyzed hydrolysis of urea was also found to throughout the study. Our ndings also indicate that the lower in
1
3
be strongly affected due to the different isotopic fractionations situ generation of CO
2
eventually signies the hydrolysis of
1
3
of substrate urea in presence of CO
together, our data clearly suggest that CO
2
environment. Taken lesser amount of C-urea in absence of adequate activation of
1
2
12
2
isotope plays a vital urease resulting from the headspace CO
Finally, we investigated the isotope-specic enzymatic
The potential effect of C-urea and C-urea on the urease activity of urease in two buffer mediums (Tris-buffer and
activation was further elucidated in response to different phosphate buffer) to eliminate the potential effect of CO on the
2
concentration.
role that facilitates the enzymatic activity of urease enzyme.
12
13
2
substrate (urea) and enzyme (urease) concentrations while pH of the reaction medium. It is worth noting that the previous
maintaining the headspace at a typical atmospheric CO2 experiments in the present study were carried out in a buffer-
free aqueous medium to avoid the effect of urease inhibition
5
in presence of buffer ions. Fig. 6 depicts that this activation in
the buffer mediums is not signicantly changed compared to
the buffer-free system. However, the diminished magnitude of
1
2
13
2 2
the concentration of the products, CO and CO observed in
the buffer mediums is exhibited to be the well-known inhibitory
effect of buffer ions in the reaction.
Conclusion
In conclusion, our ndings provide new evidences that the
catalytic activity of urease exhibits a unique isotope specic
1
2
response. We have shown that the CO
2
isotope is strongly
1
3
preferred over the CO isotope by the urease enzyme during its
catalytic activation. It was earlier reported that CO
2
11
2
acts as
a ligand in urease–urea reaction. Hence, the isotope preferential
1
2
activation of urease enzyme may be attributed to the fact that
Fig. 6 The effect of buffer medium. The hydrolysis of C-urea and
1
2
13
12
CO
CO
2
isotope possibly offers lower interaction energy than
isotope in this ligand–enzyme interaction. However,
2
C-urea in CO environment is performed in Tris buffer (pH 7.4) and
1
3
phosphate buffer (pH 7).
2
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quantum-mechanical simulations of interaction energy for both of CO
2
molecule and subsequently the concentrations of the
the isotopes would provide better insight and further explora- isotopic species were reported in parts per million (ppm).
tion of the isotope specic activation of urease enzyme. We also
provide direct experimental evidences that deepens our
Author contributions
understanding of how the activation of urease depends on the
1
2
13
isotopic compositions ( C: C) of reaction-environment as well M. P. (Manik Pradhan) arranged the funding and supervised the
as the substrate urea. Despite these new evidences, however, whole study; M. P. (Manik Pradhan) and A. M. provided the
there still remains a great deal to be explored about the conception of the study; S. M., M. P. (Mithun Pal) and A. M.
underlying mechanisms linking the specic requirement of designed the study; S. M., M. P. (Mithun Pal) and A. M. per-
1
2
CO2 isotope for the activation of urease. Moreover, new formed the experiments and analysed the data; all authors
insights into the isotope-specic responses in urease-activation draed the manuscript and critically reviewed.
are fostering exploration of a new arena to study the chemical
reactions involving metalloenzyme.
Conflict of interests
The authors declare no conict of interests.
Materials
Jack-bean urease enzyme (E.C. 3.5.1.5) was purchased from
Sigma Aldrich and C-enriched urea ( C-urea, 99%) (CCLM-
Acknowledgements
1
3
13
3
11-GMP) was obtained from Cambridge Isotopic Laborato- The authors acknowledge the funding from the Technical
ries, Inc., USA. All other chemicals, including ACS grade urea Research Centre (TRC) (No. All1/64/SNB/2014(C)) of the S. N.
1
2
12
with 99% C-enrichment ( C-urea), were acquired from Sigma Bose Centre, Kolkata for this work. S. Maithani, M. Pal and A.
Aldrich and were used without further purication. Milli-Q Maity acknowledge the Department of Science & Technology
water was used to prepare the aqueous solutions.
(DST, India) for Inspire Fellowships.
Method
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1
2
13
The aqueous solutions of C-urea, C-urea and Jack-bean
urease were utilized, unless stated otherwise, in the entire
study. The reactions were carried out in sealed round-bottomed
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asks wherein different concentrations of CO
tained. To eliminate the interference due to air, the asks were
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were main-
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1
2
13
2
2
1
2
13
(
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headspace. The headspace CO
ask and measured by a high-precision laser-based Integrated
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3
PO
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2
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
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1
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2
The measurements were made using a CO isotope analyser (CCIA
36-EP, LGR, USA) exploiting the ICOS technique as described in
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1
2
16 16
13 16 16
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Daschakraborty,
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À1
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4
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