Introduction

Alcohol Dehydrogenase (ADH), or aldehyde reductase, is an enzyme found in a variety of species ranging from Escherichia coli to Ursus arctos (Brown Bear), from Saccharomyces cerevisiae to, of course, Homo sapiens.  ADH is a member of a general classes of enzymes called oxidoreductases.  This class of enzymes utlizies the same basic mechanism to form aldehydes or ketones from an or alcohol, click here for the mechanism¹.   Note that ADH is stereospecific for which proton it remove, i.e. it removes the pro-R hydrogen.

ADH can catalyze the oxidation of many different alcohols including: primary, secondary, cyclic secondary, or hemi-acetal; in humans (especially college students) one of its major roles is in the oxidation of ethanol to acetaldehyde.   Biologically, in humans, ADH is active as a dimer.  Each subunit has two domains, a NAD binding domain (purple & green), and an alcohol (substrate) binding domain (blue & yellow). The dimer is shown to the left.

(Note: In the following section, the animation is meant to be followed in sequence, if you need to restart for any reason, click the Reload button, then Starting View below the protein image.  For best effects, let each script run to completion before starting a new one.)

Binding Domains

Click box to begin . 

Substrate Binding Domain

Pretend you are the substrate.  As we zoom in on the active site there are three amino acids  Phe 93, Leu 57, and Leu 116² that work in concert to provide the three point binding of the alcohol substrate (cyclohexonal).   This binding accounts for the stereo-specific removal of the pro-R hydrogen.

Also note the white alpha helix hinge connecting the NAD binding domain and the alcohol binding domain.

Zinc Binding Site

An integral aspect of ADH's catalysis is the electrostatic stabilization of the alcohol's oxygen by a zinc atom; this make the proton on the alcohol more acidic.  The catalytic zinc coordinates with two sulfurs from Cys 46, Cys 174 and His 67.^2,3 An ionizable water molecule occupies the 4th position on the zinc (not shown).  The water molecule is also hydrogen bonded tothe hydroxyl group of Thr-48.  The 5th and final zinc coordinate is, of course, the oxygen from the alcohol.² 

NAD Binding Domain

The NAD is bound by multiple residues off the Rossman fold, a series of beta-alpha-beta folds. Some of the residues that bind NAD include: Gly 210, Asn 225, Pro 243, Asn 242, Val 268, Asp 223, Try 178, Arg 47, Gly 292, Val 203.

(Continue to the next section without resetting the protein animation...)

Reaction Mechanism

ADH catalyzes the oxidation of alcohols by reducing NAD with a hydride.  ADH also utilizes a zinc ion to electrostatically stabilize the alcohol oxygen, thus increasing the acidity of the alcohol's proton.  In the pathway, His 51 is activated by general base catalysis such that the histidine can then accept a proton from the NAD, which in turn draws a proton from Thr 48 (may show up grey) , again demonstrating general base catalysis (although this is rather indirect since the substrate has not yet been involved).  These proton transfers ready the threonine (which is negatively charged due to proton transfer to the NAD) for accepting a proton from the alcohol of the actual substrate (cyclohexanol in the case of this particular model).  This is the first example of true base catalysis actually involving the substrate.  At the same time, since this oxidation is concerted, there is a hydride transfer to the NAD in its traditional hydride accepting region.  Thus, the whole sequence essentially amounts to a transfer of hydride to the NAD and the oxidation of an alcohol to an aldehyde.  Key points are the orientation of the amino acid proton acceptors and donors, as well as the position of the zinc ion in relation to the substrate such that it stabilizes a negative charge on the substrate thereby taking part in transition state stabilization. 

Protein Characteristics

Primary Structure:  ADH consists of two equivalent subunits with 374 residues each.  To view the sequences, click here.

Size: 80,000 g/mol. (Human ADH)

Charge: pI = 5.4

• View Acid/Base Residues: (Basic Residues - Red; Acidic Residues - Blue; Neutral - White)

Optimal pH = 8.6

Secondary Structure

• Rossman Structure - (beta-alpha-beta motif for binding NAD)
• Ramachandran Plot, click here (new window).  

The above plot of the phi/psi angles reveals that most of the amino acids in this enzyme are either in alpha helices (correlating to the blue box) or in either parallel or anti-parallel pleeted sheets (yellow and orange boxes).  Therefore, random coils are very rare in ADH.    

Hydrophobic Regions:

• Areas of high hydrophobicity (Green)
• Charged Regions (Blue)

Solvent Accessible Surface Area

• MLP Surface Plot (this will take awhile!! -- use Reload when finished viewing).

Quanta CHARMm

References:

1. ADH.  http://bio.chem.niu.edu/Chem570/Templates/ADH/

2. Alcohol Dehydrogenase.  http://florey.biosci.uq.oz.au/Html/Images/Galleria/dulley/text.html

3. Alcohol Dehydrogenase.  http://www.lmcp.jussieu.fr/iucr-top/comm/cteach/pamphlets/15/node30.html

4. Worthington Price List. Alcohol Dehydrogenase http://www.worthington-biochem.com/priceList/A/AlcoholD.html

Other Helpful Sites:

• http://www.mssc.edu/biology/B305/GTS/ss98/cjd/alcoholdh.htm
• http://www.uni-saarland.de/~mkiefer/coenz.htm