10.1002/cbic.202000185
ChemBioChem
COMMUNICATION
with substituted proteins where the normal enzyme activity is
attenuated. Reactions between dioxygen and other redox active
agents used in 2OG oxygenase catalysed reactions, e.g. DTT
likely also produce H2O2. Under our tested conditions it is notable
that TCEP did not promote the conversion of 2OG to succinate
thus, at least for some 2OG oxygenase assays, TCEP may be a
preferred reducing agent. However, the ability of TCEP to
coordinate metals ions requires consideration.[35] It is possible that
redox reactive intermediates produced at 2OG oxygenase active
sites (either in catalytically active or inactive forms) may produce
reactive oxygen species (ROS), including H2O2, that mediate the
non-enzymatic conversion of 2OG to succinate. H2O2 (or other
ROS) may also inhibit catalysis by oxidising Fe(II), either in bulk
solvent or at the active site. The extent of these processes will be
affected by the exact nature of the particular 2OG oxygenase,
including how tightly the Fe(II) ion is bound at the active site. Fe(II)
is required by 2OG oxygenases for catalysis, but as shown here,
can inhibit conversion of 2OG to succinate in the presence of L-
Asc. Moreover, high-valent enzyme-Fe complexes in 2OG
oxygenase catalysis can undergo inactivation[36] and L-Asc/DHA
undergo oxidative degradation.[22,24,26,27] Other variables include
the rate of H2O2 consumption, the presence of other metal ions,
the buffer used, and the temperature. In the latter regard it is
notable that O2 solubility in aqueous media decreases with
increases in temperature.[37] The nature of the substrate/product
may also be a factor, because substrate oxidation can be
uncoupled from that of 2OG, and the residence time of the product
on the enzyme may affect active site Fe accessibility to ROS.
The results presented here, and previously,[22,23,25,27,29-33] thus
suggest that if 2OG/O2 turnover or succinate/CO2 production are
being used for kinetic studies or inhibition assays, appropriate
controls should be implemented. The different extents to which
different redox agents promote the non-enzymatic reaction of
2OG should be considered. Our view is that the direct observation
of substrate depletion or product formation (e.g. as measured by
MS or NMR) are preferred methods for 2OG oxygenase
added as an aqueous suspension (3 µL, 45 mg/ml, 1735 units).
Sodium, potassium, magnesium, calcium, zinc, copper, nickel,
manganese, and cobalt stock solutions were made from the
corresponding chloride salts (NaCl, KCl, MgCl2, CaCl2, ZnCl2,
CuCl2, NiCl2). The Fe(II) stock solution (250 mM) was freshly
prepared using (NH4)2Fe(SO4)2·6H2O in 20 mM HCl in H2O, which
was then diluted to appropriate concentration.
Acknowledgements
We thank the Wellcome Trust, the Biotechnology and Biological
Sciences
Research
Council
(BBSRC)
(Grant
code:
BB/L000121/1) and Cancer Research UK (Grant code:
C9047/A24759) for funding our research on 2-oxoglutarate
dioxygenases.
Keywords: 2OG oxygenase • non-enzymatic • 2OG turnover 3 •
L-Ascorbate 4 • reducing agent
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Experimental section
Materials
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4
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