Importance of barrier shape in enzyme-catalyzed reactions. Vibrationally assisted hydrogen tunneling in tryptophan tryptophylquinone-dependent amine dehydrogenases

• Published in: The Journal of biological chemistry Vol. 276; no. 9; pp. 6234 - 6242
• Main Authors: Basran, J, Patel, S, Sutcliffe, M J, Scrutton, N S
• Format: Journal Article
• Language: English
• Published: United States 02.03.2001
• Subjects: Benzylamines - chemistry; Catalysis; Ethanolamine - metabolism; Indolequinones; Oxidoreductases Acting on CH-NH Group Donors - chemistry; Oxidoreductases Acting on CH-NH Group Donors - metabolism; Quinones - metabolism; Tryptamines - chemistry; Tryptophan - analogs & derivatives; Tryptophan - metabolism; Vibration
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M008141200

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.