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Alcohol Dehydrogenase

PDB ID 1htb

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1htb, resolution 2.40Å ()
Ligands: , , ,
Gene: HUMAN BETA3 CDNA (Homo sapiens)
Activity: Alcohol dehydrogenase, with EC number 1.1.1.1
Resources: FirstGlance, OCA, PDBsum, RCSB
Coordinates: save as pdb, mmCIF, xml



Overview

Alcohol dehydrogenase (PDB id 1htb), ADH, is an 80kDa enzyme that catalyzes the 4th step in the metabolism of fructose before glycolysis. In the 4th step, glyceraldehyde is converted to the glycolytic intermediate DHAP by the NADH-dependent, ADH catalyzed reduction to glycerol.[1] ADH catalyzes the oxidation of primary and secondary alcohols to their corresponding aldehydes and ketones through a mechanism that involves the removal of a hydrogen.


Structure

The initial scene () shows an overview of the molecule, allowing for a general look at the tertiary structure of alcohol dehydrogenase (it is complexed with Cl, Pyz, NAD, and Zn). A second scene () shows a close view of the ligand within each subunit. Labels have been placed on NAD, CL, and Zn to clearly establish the structure.


Within alcohol dehydrogenase, site of alcohol dehydrogenase has three important residues, Phe 93, Leu 57, and Leu 116. These three residues work together to bind to the alcohol substrate.[2]


Zn plays an important role in the catalysis. It funtions by electrostatically stabilizing the oxygen in alcohol during the reaction, which causes the alcohol to be more acidic. At the , Zinc coordinates with Cys 146, Cys 174, and His 67.[3]


NAD functions as a cosubstrate in the dehydration. NAD binds to numerous residues in a series of beta-alpha-beta folds. shows the domain where NAD binds, and many of the residues with which it interacts are selected. [4]


Alcohol dehydrogenase exists as a dimer with a zinc molecule complexed in each of the subunits. It has a SCOP catagory of an alpha and beta protein. At the N-terminal, there is a domain that is all beta; however, the C-Terminal domain is alpha and beta, so the catagory is alpha and beta. The C-Terminal core has 3 layers of alpha/beta/alpha and parallel beta sheets of 6 strands.[5]


Reaction and Mechanism

In the oxidation mechanism, ADH is momentarily associated with nicontinamide adenine dinucleotide (NAD+), which functions as a cosubstrate. In its reaction, alcohol dehydrogenase uses zinc and NAD to facilitate the reaction. The function of zinc is to position the –OH group on the ethanol in a conformation that allows for the oxidation to occur. NAD then acts as a cosubstrate and performs the oxidation.

[6] The of alcohol dehydrogenase reaction is as follows: CH3CH2OH + NAD+ -> CH3COH (acetaldehyde) + NADH + H+ (Note: The reaction is actually reversible although the arrow does not show it) [7] The step-wise reduction mechanism for ADH is shown on the left. In the mechanism, His 51 is deprotonated and activated by a base catalyst. This allows histidine to accept a proton from NAD, which also draws a proton Thr 48. As a result of the proton transfer, the Thr is prepared to accept a proton from the alcohol substrate. While Thr accepts the proton, there is also a hydride transfer to NAD. The whole process can be summarized as the oxidation of an alcohol to an aldehyde in concert with the transfer of a hydride to NAD.[8]


The Mechanism for alcohol dehydrogenase follows an random bisubstrate mechanism.[9] In the mechanism, the NAD+ and alcohol bind to the enzyme, so that the enzyme is now attached to the two subtrates. While attached, the hydrogen is formally transferred from the alcohol to NAD, resulting in the products NADH and a ketone or aldehyde. The two products are then released, and the enzyme has catalyzed the reaction.


Kinetics

The alcohol dehydrogenase catalyzed aldehyde-NADH reaction show kinetics consistent with a random-order mechanism, and the rate-limiting step is the dissociation of the product enzyme-NAD+ complex. [10] Alcohol dehydrogenase is more effective for smaller alcohol substrates, and it becomes less effective as substrate size increases. It is also more effective for primary than secondary alcohols.[11] In a study where ADH was immobilized in tresyl-chloride-activate agarose, it was shown that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate.[12]


Regulation

Substrate size is a regulator, where larger substrates inhibit alcohol dehydrogenase. Further, alcohol dehydrogenase is somewhat inhibited if the substrate is a secondary alcohol, as opposed to a primary alcohol. [13] Pyrazoles have also been shown to be inhibitors of ADH. [14] Other inhibitors include heavy metals, thiourea, purine and pyrimidine derivatives, and both chloroethanol and flouroethanol. [15] Activators include sulfhydryl activating reagents, mercaptoethanol, dithiothreitol, and cysteine.[16]


References

  1. ↑ Voet, et. al. Fundamentals of Biochemistry: 3rd Edition. Hoboken: Wiley & Sons, Inc, 2008.
  2. ↑ Protein: Alcohol Dehydrogenase. The College of Saint Benedict and Saint John's University. 1 March 2010 < http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcoholdehydro/index.htm>
  3. ↑ Protein: Alcohol Dehydrogenase. The College of Saint Benedict and Saint John's University. 1 March 2010 < http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcoholdehydro/index.htm>
  4. ↑ Protein: Alcohol Dehydrogenase. The College of Saint Benedict and Saint John's University. 1 March 2010 < http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcoholdehydro/index.htm>
  5. ↑ Protein: Alcohol dehydrogenase from Human (Homo sapiens), different isozymes. SCOP. 2009. 1 March 2010 < http://scop.berkeley.edu/data/scop.b.d.c.b.b.c.html>
  6. ↑ Protein: Alcohol Dehydrogenase. The College of Saint Benedict and Saint John's University. 1 March 2010 < http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcoholdehydro/index.htm>
  7. ↑ Voet, et. al. Fundamentals of Biochemistry: 3rd Edition. Hoboken: Wiley & Sons, Inc, 2008.
  8. ↑ Protein: Alcohol Dehydrogenase. The College of Saint Benedict and Saint John's University. 1 March 2010 < http://www.users.csbsju.edu/~hjakubow/classes/rasmolchime/99ch331proj/alcoholdehydro/index.htm>
  9. ↑ Voet, et. al. Fundamentals of Biochemistry: 3rd Edition. Hoboken: Wiley & Sons, Inc, 2008.
  10. ↑ Dickinson FM, Monger GP. A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates. Biochem J. 1973 Feb;131(2):261-70. PMID:4352908
  11. ↑ Dickinson FM, Monger GP. A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates. Biochem J. 1973 Feb;131(2):261-70. PMID:4352908
  12. ↑ Bille V, Remacle J. Simple-kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl-chloride-activated agarose. Eur J Biochem. 1986 Oct 15;160(2):343-8. PMID:3769934
  13. ↑ Dickinson FM, Monger GP. A study of the kinetics and mechanism of yeast alcohol dehydrogenase with a variety of substrates. Biochem J. 1973 Feb;131(2):261-70. PMID:4352908
  14. ↑ Blomstrand R, Ostling-Wintzell H, Lof A, McMartin K, Tolf BR, Hedstrom KG. Pyrazoles as inhibitors of alcohol oxidation and as important tools in alcohol research: an approach to therapy against methanol poisoning. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3499-503. PMID:115004
  15. ↑ Alcohol Dehydrogenase. Worthington Biochemical Corporation . 31 March 2010 < http://http://www.worthington-biochem.com/ADH/default.html>
  16. ↑ Alcohol Dehydrogenase.Worthington Biochemical Corporation . 31 March 2010 < http://http://www.worthington-biochem.com/ADH/default.html>

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