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Aromatic Amine Dehydrogenase and Long Distance Proton Transfer

Page history last edited by Henry Warrington 12 years, 6 months ago



Aromatic Amine Dehydrogenase (AADH) is a molecule that catalyzes a reaction which converts and amine group to an aldehyde group on certain aromatic compounds.  This reaction has been tracked by X-ray crystallography to bring to light how this occurs.  It has been found that a proton transfer occurs, despite a large energy barrier for this type of reaction.  This is allowed due to the proton's quantum tunneling.


Information about AADH as a molecule:


AADH is a protein that catalyzes the breakdown of several amino acids.  AADH's are inducible proteins that reside in the periplasm of some Gram-negative bacteria.3  The periplasm is a section that exists between the two cell membrane sections of Gram-negative bacteria.  It has a molecular weight of 108,900 amu, over double that of the average protein!  It is isoelectric at pH 5.2, meaning it is slightly acidic. It remains active up to 60 degrees Celsius, and it contains a prosthetic Tryptophan Tryptophylquinone (TTQ) group, which is what allows it to react.This TTQ group is covalently bonded to the AADH protein as a cofactor (a molecule necessary in order for the protein to be in an active state).3


Fig 1. Structure of TTQ molecule, "Protein" represents where it is bound to AADH


The TTQ group allows AADH to act as an enzyme that catalyzes the an oxidation reaction.  This reaction takes in a primary aromatic amine group, water, and an electron acceptor (Which varies based on the bacteria in question) and gives off an aldehyde group, ammonia and a reduced form of the electron acceptor.2  First, TTQ is reduced by the primary amine group, making the aldehyde, then the electron acceptor reoxidized the TTQ group, allowing the protein to continue catalyzing the reaction.  This allows the one enzyme to continually break down amino acids so that the bacteria can use them as a source of carbon and nitrogen3


For a full curved arrow mechanism for this reduction reaction, click here: http://www.jbc.org/content/282/33/23766/F1.expansion.html3


AADH depends on TTQ  and it catalyzes proton transfer by quantum mechanical tunneling. It oxidatively deaminates aromatic primary amines to form aldehydes. The electrons releases are transferred from AADH to the electron acceptor and moves from there to the respiratory chain.4 A recent computation study of proton tunneling in AADH reveals that with tryptamine, the degree of tunneling is calculated to exceed 99.9%.5 There are been studies that show that vibrationally assisted tunneling is the most likely mechanism of bond cleavage for the reaction of AADH with benzylamine.6


Kinetic Isotope Effect:


The Kinetic Isotope Effect is the ratio of the rates of reaction between when a reaction is carried out with a heavier isotope and when it is not. The KIE is denoted by the ratio of the rates of reaction for the original molecule and the isotope (kH/kD). The KIE is carried out usually by replacing Hydrogen with Deuterium. This ratio for the replacing of Hydrogen with Deuterium is usually between 1-8 for most isotopes. The higher the ratio the greater the rate of the reaction of the original molecule over the rate of the isotope. Also heavier isotopes tend to form stronger bonds with carbon chains so the higher the KIE indicates the number of R-H that are broken in the reaction mechanism.7


Proton Tunneling and KIE in the AADH Reaction:


The reaction catalyzed by AADH is similar to a one dimensional particle in a box with a finite potential energy barrier on one side. The way that the reaction takes place via tunneling is based on the de Broglie wavelength of the Hydrogen and its isotopes. As the reaction climbs up the potential energy barrier in order to proceed, it eventually reaches a point where the barrier width between reactant and product is less than the de Broglie wavelength of the Hydrogen atom or isotope so the hydrogen atom tunnels through the barrier and the reaction proceeds. Since Hydrogen is lighter than Deuterium, it has a larger wavelength which is why reactions with Hydrogen tend to be faster than those with its isotopes. As shown in Fig 2 the Hydrogen atom has a much larger wavelength than Deuterium or Tritium so it is able to tunnel through the potential energy barrier into the adjacent well much sooner than its isotopes. This causes the reaction to move much faster than normal because it can tunnel long before the reaction reaches the activation energy.8 


Fig. 2 How a proton or proton isotope tunnels through a potential energy barrier


In AADH the KIE ratio between Hydrogen and Deuterium is about 55, which is much larger than that of the regular KIE. Since the KIE ratio is so high, it can be determined that the reaction mechanism has many R-H bonds broken as shown in the mechanism above. Since there are so many R-H bonds to break proton transfer helps to speed the reaction up so because the reaction does not have to reach the activation energy every time to break an R-H bond.9


Other enzymes that use tunneling:


     AADH isn’t the only biological process that uses quantum tunneling. The coenzyme "B12-dependent methylmalonyl-CoA mutase" also uses it for proton transfer reactions. It shows a very large kinetic isotope effect which indicates that it also proceeds by a highly quantum tunneling mechanism.  However B12-depedent molecules that have similar reactions don't have the same mechanism as AADH. They are believed to operate by radical translocation which is the relocation of the radical site by intermolecular abstraction of hydrogen or a side chain on the molecule. The observation of very large hydrogen to deuterium (H/D) KIEs for hydrogen radical transfer in several enzymes raises the possibility that tunneling may be a common strategy for the proteins reaction.10

     In bonds, the atoms on each end vibrate in and out as shown in Fig 3 below.  In the case of classical physics, the energy barrier would be far too high for the reaction to occur, however, quantum tunneling allows it to occur.  The proton has to travel over such a long distance, around other molecules, it would never happen classically.  However, due to quantum tunneling, the proton is able to pass through the other molecules directly between it and its final destination.


Fig 3. A diagram of the quantum oscillator model, areas outside the parabola indicate quantum tunneling.




Fig. 3 A picture of Methylmalonyl coenzyme A.



1. Govindaraj, Shanthi, Edward Eisenstein, Limei H. Jones, Joann Sanders-Loehr, Andrei Y. Chistoserdov, Victor L. Davidson, and Steven L. Edwards. "Aromatic Amine Dehydrogenase, a Second Tryptophan Tryptophylquinone Enzyme." Journal of Bacteriology 176.10 May (1994): 2922-29. Web. 25 Oct. 2011.


2. Iwaki, Masayoshi, Toshiharu Yagi, Kihachiro Horike, Yikikazu Saeki, Tsutomu Ushijima, and Mitsuhiro Nozak. "Crystallization and Properties of Aromatic Amine Dehyrdogenase from Pseudomonas sp." Archives of Biochemistry and Biophysics 220.1 Jan. (1983): 253-62. Web. 25 Oct. 2011.


3. Roujeinikova, Anna, Parvinder Hothi, Laura Masgrau, Michael J. Sutcliffe, Nigel S. Scrutton, and David Leys. "New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates." The Journals of Biological Chemistry 28217 Aug. (2007): 23766-77. Web. 29 Oct. 2011.


4. Roujeinikova Anna, Parvinder Hothi, Laura Masgrau, Michael J. Sutcliffe, Nigel S. Scrutton, and David Leys, Linus O. Johannissen, Jaswir Basran, Kara E. Ranaghan, Adrian J. Mulholland. "Atomic Descriptions of an Enzyme Reaction Dominated by Proton Tunneling."  Science April 2006.


5. Roujeinikova Anna, Parvinder Hothi, Laura Masgrau, Michael J. Sutcliffe, Nigel S. Scrutton, and David Leys, Linus O. Johannissen, Jaswir Basran, Kara E. Ranaghan, Adrian J. Mulholland. "Hydrogen tunneling in enzyme-catalysed H-transfer reaction: flavoprotein and quinoprotein systems. Phil. Trans. R. Soc. B Aug. 2006.


6. Jaswir Basran, Shila Patel, Michael J. Sutcliffe, Nigel S. Scrutton. "Importance of Barrier Shape in Enzyme-catalyzed Reactions" The Journals of Biological Chemistry  March 2001.


7. Kinetic Isotope Effects, University of California, Davis, UCDavis Chemwiki,  October 30, 2011, < http://chemwiki.ucdavis.edu/Physical_Chemistry/Quantum_Mechanics/Kinetic_Isotope_Effect>


8. Amnon Kohen, Judith P Klinman, Hydrogen Tunneling in Biology, University of Iowa, July 1999, Octover 20, 2011, <http://www.chem.uiowa.edu/faculty/kohen/group/Journal/C&B99K&K.pdf>


9. Masgrau L,Ranaghan KE,Scrutton NS,Mulholland AJ,Sutcliffe MJ, Tunneling and classical paths for proton transfer in an enzyme reaction dominated by tunneling: oxidation of tryptamine by aromatic amine dehydrogenase, PubMed.cov, March 22, 2007, October 30, 2011, < http://www.ncbi.nlm.nih.gov/pubmed/17388439>


10. Agnieszka Dybala-Defratyka, Piotr Paneth, Ruma Banerjee, Donald G. Truhlar. "Coupling of hydrogenic tunneling to active-site motion in the hydrogen radical transfer catalyzed by a coenzyme B12-dependent mutase." Proceedings of National Academy of Sciences May 2007.



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