Importance of barrier shape in enzyme-catalyzed reactions. Vibrationally assisted hydrogen tunneling in tryptophan tryptophylquinone-dependent amine dehydrogenases.
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 degrees 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.
Basran
J
J
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
Patel
S
S
Sutcliffe
M J
MJ
Scrutton
N S
NS
D001596:Benzylamines / Q000737:chemistry; D002384:Catalysis; D019856:Ethanolamine / Q000378:metabolism; D045563:Indolequinones; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000378:metabolism; D011809:Quinones / Q000378:metabolism*; D014363:Tryptamines / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism*; D014732:Vibration
D001596:Benzylamines; D045563:Indolequinones; D011809:Quinones; D014363:Tryptamines; C070569:tryptophan tryptophylquinone; C030820:tryptamine; D019856:Ethanolamine; D014364:Tryptophan; C030796:benzylamine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037193:aromatic amine dehydrogenase