Importance of Barrier Shape in Enzyme-catalyzed Reactions

Abstract

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 Htransfer in MADH and AADH with fast and slow substrates and highlight the importance of barrier shape in determining reaction rate. Aromatic amine dehydrogenase (AADH)1 and methylamine dehydrogenase (MADH) catalyze the oxidative deamination of primary amines to their corresponding aldehydes and ammonia.The physiological electron acceptors of MADH are amicyanin (for the Paracoccus denitrificans enzyme; Refs. 1-3) or a c-type cytochrome (for the Methylophilus methylotrophus enzyme; Ref. 4). AADH transfers electrons (derived from the deamination of aromatic amines) to the copper protein azurin (5). In a reductive half-reaction, both enzymes reduce the prosthetic group tryptophan tryptophylquinone (TTQ) through the initial formation of a carbinolamine intermediate ( Fig. 1; Refs. 5 and 6); loss of water from this carbinolamine produces an iminoquinone. The iminoquinone decays by breakage of the substrate C-H bond. An active site base that abstracts a proton from the substrate initiates bond cleavage (7). This step in the reductive half-reaction is rate-limiting, as demonstrated by the unusually large kinetic isotope effect (KIE) observed with methylamine (MADH; Refs. 8 -10) and dopamine (AADH; Ref. 11).Previously we demonstrated that cleavage of the substrate C-H bond of M. methylotrophus (sp. W 3 A 1 ) MADH is associated with a large KIE (16.8 Ϯ 0.5) and that the KIE is independent of temperature (8). Through detailed temperature dependence studies, we were able to suggest that C-H bond breakage occurs by extreme quantum tunneling (8). Quantum tunneling effects in enzymatic hydrogen transfer have been observed in only a small number of enzyme systems (8, 12-21). Most have been modeled using the classical formulations of transition state theory, incorporating a tunne...

Interpretation of Kinetic Isotope Effects in Enzymatic Cleavage of Carbon-Hydrogen Bonds

“…Much the same holds for the data reported for a number of AADH reactions [12]. However, the MADH reaction in which methylamine is replaced by ethanolamine [12] is an exception.…”

“…Scrutton and coworkers [11,12] have studied the oxidative demethylation of primary amines to form aldehydes and ammonia by methylamine dehydrogenase (MADH) and also by aromatic amine dehydrogenase (AADH), which shows much the same behavior. In these reactions a proton is abstracted by an active-site base from an iminoquinone intermediate while an electron is transferred to a tryptophylquinone cofactor.…”

“…However, the MADH reaction in which methylamine is replaced by ethanolamine [12] is an exception. The latter reaction exhibits the same KIE as the methylamine reaction, implying the same range of z(298) values.…”

“…If the data pass this test, qualitative and quantitative information can be obtained on the mechanism of the tunneling reaction and the structure of the reaction center. In subsequent sections the model is applied to a representative set of kinetic data on enzymatic systems and models thereof, including soybean lipoxygenase-1 (SLO1) [5,6], coenzyme B 12 [9,10], primary amine dehydrogenases [11,12], and phenylalanine hydroxylase [13].…”

Optimizing the Michaelis Complex of Trimethylamine Dehydrogenase

“…The mechanism of amine oxidation catalyzed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with topaquinone or tryptophan tryptophylquinone redox centers (1,2). In some cases C-H bond cleavage by these enzymes has been shown to involve quantum tunneling mechanisms for transfer of the H nucleus to a base in the enzyme active site (3)(4)(5). By contrast, the mechanism of amine oxidation by flavoproteins is understood less well.…”