a-lytic protease4) no kinetic model was developed to explain the
findings. A short (,1 second) and product dependent lag-phase
has been identified with the enzyme soybean lipoxygenase-1
(SML-1).2,3,18,19 A kinetic model was developed for this system
that included a reactive (FeIII) and non-reactive (FeII) enzyme and
could fit data with lag-phase of up to 1 second. We have attempted
to develop such a model for the findings reported here but models
that could lead to reasonable fit to the time course of the FAD
reduction (Fig. 1) could not explain the dUMP concentration
dependency (Fig. 2). Such a kinetic model is described in the
Electronic Supplementary Information{ and attempts to develop a
more comprehensive models are under way. We hope that the data
presented here will lead to more theoretical work in the field of
delayed reactivity.
dUMP dependent lag-phase will redirect the thinking on FDTS
mechanism, and will indicate how FDTS enzymes might be
inhibited in a way that will not affect human or other classical TSs.
As a general note, the substrate dependent lag-phase reported
here might be more general than realized hereto. In most cases it is
not easy or even possible to follow the preliminary effect of one
substrate in a multi-substrate reaction. Such phenomenon might
be hidden in other systems either due to mixing effects or because
the substrate concentration was above Kf. Consequently, it might
be interesting to search for it in cases where different experimental
settings suggest different orders of binding under different
conditions.
Notes and references
As for the dUMP binding site, it might be bound at its reactive
site and react with CH2H4folate after this second substrate binds
the reduced enzyme, or it might be bound at a different site (an
allosteric site or another dUMP binding site close to a different
flavin in this tetrameric enzyme). Following the reduction of FAD
and the release of NADP+, the dUMP may react with
CH2H4folate (in the first scenario), or a second dUMP may need
to bind the reduced active site between the flavin and the
nucleophilic Ser8 (in the second case). Future trapping experiments
with labeled dUMP using quench-flow methodology may resolve
this question.
1 A. Fersht, Structure and Mechanism in Protein Sciences: A Guide to
Enzyme Catalysis and Protein Folding, W. H. Freeman, New York,
1998.
2 H. Berry, H. De´bat and V. L. Garde, J. Biol. Chem., 1998, 273,
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3 M. J. Knapp and J. P. Klinman, Biochemistry, 2003, 42, 11466–11475.
4 K. C. Haddad, J. L. Sudmeier, D. A. Bachovchin and
W. W. Bachovchin, Proc. Natl. Acad. Sci. U. S. A., 2005, 102,
1006–1011 and personal communication with B. Bachovchin.
5 C. W. Carreras and D. V. Santi, Annu. Rev. Biochem., 1995, 64,
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6 R. M. Stroud and J. S. Finer-Moore, Biochemistry, 2003, 42, 239–247
and many references cited therein.
In summary, Liebl and co-workers10 suggested a mechanism for
the conversion of dUMP to dTMP catalyzed by FDTS. Their
mechanism implied that dUMP binds to the FDTS with FAD in
the oxidized state followed by NADPH binding and FAD
reduction. Their main supportive evidence was the enhanced
oxidation of NADPH by molecular oxygen in the presence of
dUMP (followed by 340 nm absorbance reduction as the NADPH
converted to NADP+). McClarty and co-workers9 on the other
hand, used tritium-release kinetics (single point analyzed after a
short reaction period using [5-3H]dUMP) suggested a Ping Pong
mechanism in which NADPH is oxidized and NADP+ leaves prior
to CH2H4folate binding, H4folate release, dUMP binding and
dTMP release. These experiments and findings appeared contra-
dictory at first. Our finding of a dUMP dependent lag-phase can
explain some of these different observations. The dUMP enhances
the NADPH oxidation by deleting the lag-phase and thus
appeared to bind first in Liebl’s experiments. Its reactive binding
as acceptor of the methylene (either from the CH2H4folate, or
enzymatic methylene intermediate) may be of a different nature as
suggested by the binding constant and energetics of dUMP as
activator vs. its binding as a substrate. Such a mechanism could
rationalize the different binding and release pattern suggested by
McClarty and co-workers.9 We believe that the new finding of a
7 H. Myllykallio, G. Lipowski, D. Leduc, J. Filee, P. Forterre and
U. Liebl, Science, 2002, 297, 105–107.
8 I. I. Mathews, A. M. Deacon, J. M. Canaves, D. McMullan,
S. A. Lesley, S. Agarwalla and P. Kuhn, Structure, 2003, 11, 677–690.
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10 S. Graziani, Y. Xia, J. R. Gurnon, J. L. Van Etten, D. Leduc,
S. Skouloubris, H. Myllykallio and U. Liebl, J. Biol. Chem., 2004, 279,
54340–54347.
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15 N. Agrawal, S. A. Lesley, P. Kuhn and A. Kohen, manuscript in
preparation.
16 Curve fitting was carried out as a least root-mean-square, standard
deviations weighted, non-linear regression with the software
Kaleidagraph1.
17 Calculated using DGu = 2RTln[KMh/kbT], where R is the gas constant,
T is the absolute temperature, h is Planck’s constant and kb is the
Boltzmann constant.
18 M. J. Schilstra, G. A. Veldink, J. Verhagen and J. F. G. Vliegenthart,
Biochemistry, 1992, 31, 7692–7699.
19 M. J. Schilstra, G. A. Veldink and J. F. G. Vliegenthart, Biochemistry,
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