Case 13 Inhibition of Alcohol Dehydrogenase Focus concept The inhibition of the

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Case 13 Inhibition of Alcohol Dehydrogenase Focus concept The inhibition of the alcohol dehydrogemase by a formamide compound is examined Prerequisites Principles of enzyme kinetics Idenification of inhibition via Lineweaver-Bark plots Background Alcohol dchydrogenase (ADiH) is the enayme that is responsibke for converting cthanol to acetakdehyde (the reaction is shown in Figure 13,1), It is the enzyme responsible for the metabolism of ethanol in the alcoholic beverages we consume. Five different isozymes of ADH have been identifiod, and i has been shown that the cnuayme has a rather broad substrate specificity and can oxidizne akdchydes as well as primary and secondary akcohols. For example, ADH ean also oxidize methanol (wood akobol) and ethylene alcohol (antifreeze). The poisonous nature of these compounds results from the ADH-catalyzed conversion of these compounds to toxic products. For example, ADH converts methanol to formakdehyde, which is toxic to the optic nerve and can produce blindness In high doses, formaldehyde may be fatal In this study, the authors investigated the abiliny of formamide compounds to inhibit alcohol dehydrogenase. Only a portion of their data is presented here. The authors were able to propose a mechanism for the inhibtion from the emensive data they collected using a wide varicty of formamide compounds. The mechanism is shown in Figure 13.2 NAH H, O ADH Ethand NAD Figure 13.1:AD-catalyzed oxidation of ethanol. alcohol NAD E-NADE-NAD -alcohol NADHl E-NADH- Figure 13.2: Mechanism of ADH1. The inhibitor binds as an aldehyde analog.

Explanation / Answer

Pyrazole is a competitive inhibitor. It decreases the apparent Km. In comparative studies, reactions with and without pyrazole give a common y-intercept which is indicative of a competitive inhibitor. This makes it less effective in studies and in alleviating poisoning effect when substrate alcohol concentration is increased.

In studies, ADH-NAD-pyrazole complexes have been crystallized and pyrazole has been found to occupy the active alcohol binding site and form a bridge between Zn atoms of ADH and nicotinamide moieties of the coenzyme.

Formamides are different from pyrazoles.

Formamides are unreactive analogues of the aldehyde substrates of alcohol dehydrogenases.

Formamide inhibits alcohol dehydrogenase (ADH) acting as a uncompetitive inhibitor.  

X-ray crystallography study of alcohol inhibitor has revealed at 1.6 Å resolution, the puckering of the reduced nicotinamide ring of NADH in michaelis complex of a dehydrogenase with NADH and a substrate or substrate analogue, (R)-N-1-methylhexylformamide.

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