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J. Biol. Chem., Vol. 276, Issue 9, 6234-6242, March 2, 2001
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From the C-H bond breakage by tryptophan
tryptophylquinone (TTQ)-dependent methylamine dehydrogenase
(MADH) occurs by vibrationally assisted tunneling (Basran, J.,
Sutcliffe, M. J., and Scrutton, N. S. (1999)
Biochemistry 38, 3218-3222). We show here a similar mechanism in TTQ-dependent aromatic amine dehydrogenase (AADH). The
rate of TTQ reduction by dopamine in AADH has a large, temperature independent kinetic isotope effect (KIE = 12.9 ± 0.2), which
is highly suggestive of vibrationally assisted tunneling. H-transfer is
compromised with benzylamine as substrate and the KIE is deflated (4.8 ± 0.2). The KIE is temperature-independent, but reaction rates are strongly dependent on temperature. With tryptamine as substrate reaction rates can be determined only at low temperature as
C-H bond cleavage is rapid, and an exceptionally large KIE (54.7 ± 1.0) is observed. Studies with deuterated tryptamine suggest vibrationally assisted tunneling is the mechanism of deuterium and, by
inference, hydrogen transfer. Bond cleavage by MADH using a slow
substrate (ethanolamine) occurs with an inflated KIE (14.7 ± 0.2 at 25 °C). The KIE is temperature-dependent, consistent with differential tunneling of protium and deuterium. Our observations illustrate the different modes of H-transfer in MADH and AADH with fast
and slow substrates and highlight the importance of barrier shape in
determining reaction rate.
Importance of Barrier Shape in Enzyme-catalyzed
Reactions
VIBRATIONALLY ASSISTED HYDROGEN TUNNELING IN TRYPTOPHAN
TRYPTOPHYLQUINONE-DEPENDENT AMINE DEHYDROGENASES*
§,§,
Department of Biochemistry and the
¶ Department of Chemistry, University of Leicester, University
Road, Leicester LE1 7RH, United Kingdom
*
This work was funded by grants from the Biotechnology and
Biological Sciences Research Council and the Lister Institute of Preventive Medicine.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
A Lister Institute research professor. To whom
correspondence should be addressed. Tel.: 44-116-223-1337; Fax:
44-116-252-3369; E-mail, nss4@le.ac.uk.
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