A kinetic study of the rat liver adenosine kinase reverse reaction

Article abstract of DOI:10.1080/15257770802146544

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH3. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels. Copyright Taylor & Francis Group, LLC.

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A Kinetic Study of the Rat Liver
Adenosine Kinase Reverse Reaction
a
a
a
a
a
D. Vannoni , S. Giglioni , A. Santoro , E. Aceto , E. Marinello
a
&
a
R. Leoncini
Dipartimento di Medicina Interna, Sc. Endocrino-Metaboliche e
Biochimica , Università di Siena , Italy
Published online: 03 Jul 2008.
To cite this article: D. Vannoni , S. Giglioni , A. Santoro , E. Aceto , E. Marinello & R. Leoncini (2008)
A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction, Nucleosides, Nucleotides and
Nucleic Acids, 27:6-7, 872-875, DOI: 10.1080/15257770802146544
To link to this article: http://dx.doi.org/10.1080/15257770802146544
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Nucleosides, Nucleotides, and Nucleic Acids, 27:872–875, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802146544
A KINETIC STUDY OF THE RAT LIVER ADENOSINE KINASE
REVERSE REACTION
D. Vannoni, S. Giglioni, A. Santoro, E. Aceto, E. Marinello, and R. Leoncini
Dipartimento di Medicina Interna, Sc. Endocrino-Metaboliche e Biochimica, Universit a` di
Siena, Italy
2
Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We
studied some biochemical properties not hitherto investigated and demonstrated that the reaction can
be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine
and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH
3
. The exoergonic
ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This
reaction could be used by cells under particular conditions of energy deficiency and, together with
myokinase activity, may help to restore physiological ATP levels.
Keywords Adenosine kinase; rat liver; adenosine; purine metabolism
INTRODUCTION
Many reports indicate that adenosine plays an important role in regulating
a variety of metabolic processes. Adenosine may act as a “local hormone”
in mammalian tissues showing a diversity of effects, including modulation
of blood flow, inflammation, neurotransmission, pain and the endogenous
response to ischemia.[ Most basal adenosine production during normoxia
1]
[2]
is derived from the action of S-adenosylhomocysteine hydrolase. However,
during enhanced oxygen demand or metabolic load, increased amounts of
ꢁ
adenosine are formed almost exclusively from AMP by 5 -nucleotidase. In
cells, the adenosine generated may be deaminated to inosine by adenosine
deaminase (ADA), phosphorylated to AMP by adenosine kinase (AdK), or
transported into extracellular fluid where it acts by coupling to A1, A2, or A3
[3]
receptors. Comparison of the kinetic properties of AdK and ADA suggests
that under normal conditions most intracellular adenosine is metabolized
by AdK.
The latter enzyme has been studied from several points of view: it
has been purified to homogeneity from various mammalian tissues, has a
Address corresopndence to Daniela Vannoni, Department of Internal Medicine, Endocrine
Metabolic Science and Biochemistry, Siena University, Siena, Italy. E-mail: vannoni@unisi.it
8
72

D. Vannoni et al.
873
monomeric structure and a MW estimated around 40,000;[4] 2it5s,6k]inetic prop-
[
erties, gene expression, and structure have been described.
In this study we analyzed the reversibility of the AdK reaction and
demonstrated that it can be detected only if coupled with the ADA reaction.
This series of reactions transforms adenosine into inosine + NH according
3
to Scheme 1
MATERIALS AND METHODS
AdK was purified to homogeneity from rat liver according to the method
of Yamada et al.[ with slight modifications. The purified enzyme showed
specific activity, Vmax and Km for substrates of the same order of magnitude
as reported in the literature.[ The assay mixture used to test AdK reverse
activity contained: 25 mM Bis-Tris (pH 6.5), 1 mM MgCl 1.2 mM AMP, 1.2
7]
7]
2,
mM ADP, 0.9 U commercial ADA (from Sigma), 1.4 µg purified rat liver
AdK and H O to a final volume of 0.125 ml. The mixture was incubated at
2
◦
3
7 C for 20 minutes, deproteinized by adding concentrate perchloric acid
(
2.5µl), and neutralized with 5N KOH. The reaction products were analyzed
by HPLC. :
An adenosine peak was not detectable in chromatograms, demonstrat-
ing the complete conversion into inosine by the ADA coupled reaction. Ac-
tivity of the reverse reaction was calculated by the combined areas under
the ATP and inosine peaks, compared to a calibration curve processed un-
der the same conditions.
Time course, linearity with protein concentrations, effect of alternative
substrates and salts, and Km for ADP and other alternative substrates of the
reaction were also determined.
RESULTS
The reaction was linear with respect to time (up to 60 minutes) and pro-
tein concentration. Activity peaked at pH 6.5 and fell away sharply below pH
5
and above pH 8. The rate of the reverse reaction, estimated in the optimal
SCHEME 1

8
74
Adenosine Kinase Reverse Reaction in Rat Liver
TABLE 1 Specific activity (S.A.) and corresponding Km values of Reaction 1 calculated using
ADP and alternative substrates. Values are the means of five experiments ± St. Dev
Substrates
S .A. ± St Dev mmol/ (min*mg) %Activity with respect to ADP
Km (mM)
ADP
GDP
dGDP
dADP
0.815 ± 0.045
0.322 ± 0.018
0.225 ± 0.011
0.185 ± 0.008
100
41
28
0.108 ± 0.005
0.216 ± 0.009
0.503 ± 0.028
1.970 ± 0.053
23
conditions (described in the Materials and Methods section), resulted about
6
times lower than the forward.
Table 1 reports the enzyme specific activity assayed using 0.2 mM ADP,
compared to alternative substrates; the respective K m values were calcu-
lated using substrate concentrations ranging between 0.05 to 5 mM. Activity
and substrate affinity were greatest for ADP, but the reaction was also effec-
tive with GDP, dADP and dGDP. Conversely, pyrimidine compounds (CDP
and UDP) were not good substrates. Table 2 shows that MgCl was the best
2
cofactor for the reaction.
DISCUSSION
In this paper we studied the reaction AMP + ADP →Ado + ATP, cat-
alyzed by rat liver AdK, which is the reverse of the original AdK reaction.
According to the literature, this reaction is strongly directed towards the
production of adenosine and ATP for energy reasons, but we found that the
energetic gap can be filled by the coupled ADA reaction which transforms
Ado into inosine and ammonia. This second reaction is exergonic with a
0
8
ꢀ
G of -4 to -10 kcal/mol , has a low K for substrate and high efficiency,
m
close to diffusion-limited, and occurs in most mammalian tissues. Under our
conditions, the equilibrium is shifted towards ATP synthesis.
Further experiments are needed to determine whether this reverse
reaction has physiological significance and could play a role in particu-
lar situations associated with ATP deficiency or increased ATP demand,
TABLE 2 Activity of Reaction 1 assayed with
alternative salts at the same concentrations as
MgCl2(1.2 mM), expressed as a percentage
with respect to MgCl2
MgCl2
CoCl2
MgSO4
MnCl2
ZnCl2
CaCl2
100%
95%
90%
77%
13%
4%

D. Vannoni et al.
875
as during prolonged muscle work (when ATP is dramatically reduced),
fructose-induced hyperuricemia with ATP depletion, situations of severe nu-
cleotide depletion (such as rheumatoid arthritis, anemia, and cell division)
and transient ischemia-anoxia.
REFERENCES
1
.
.
Sakowicz, M.; Grden, M.; Pawelczyk, T. Expression level of adenosine kinase in rat tissues. Lack of
phosphate effect on the enzyme activity, Acta Biochim. Pol. 2001, 48, 745–754.
Lloyd, H.G.; Deussen, A.; Wuppermann, H.; Schrader, J. The transmethylation pathway as a source
for adenosine in the isolated guinea-pig heart. Biochem. J. 1988, 252, 489–494.
Stiles, G.L. Adenosine receptors. J. Biol. Chem. 1992, 267, 6451–6454.
2
3
4
.
.
Yamada, Y.; Goto, H.; Ogasawara, N., Differences of adenosine kinases from various mammalian
tissues. Comp. Biochem. Physiol. B. 1982, 71, 367–72.
5.
6.
7.
8.
Moffatt, B.A.; Wang, L.; Allen, M.S.; Stevens, Y.Y.; Qin, W.; Snider, J.; von Schwartzenberg, K., Adeno-
sine kinase of arabidopsis.Kinetic properties and gene expression, Plant Physiol. 2000, 124, 1775–85.
Vonrhein, C. Bonisch, H.; Schafer, G.; Schulz, G.E. The structure of a trimeric archaeal adenylate
kinase, J., Mol., Biol. 1998, 282, 167–179.
Yamada, Y.; Goto, H.; Ogasawara, N. Adenosine kinase from human liver. Biochim. Biophys. Acta ,1981,
660(1), 36–43.
Ma, P.F.; Fisher, J.R. Comparative studies of mammalian adenosine deaminases: some distinctive
properties in higher mammals. Comp. Biochem. Physiol. 1969, 31, 771–781.