Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with diazonium-1H-tetrazole.

The research studied the effect of modifying the cysteine residue 174 in horse liver alcohol dehydrogenase using diazonium-1H-tetrazole, finding that it inactivates the enzyme. The research highlighted the importance of this specific cysteine residue for enzyme functionality.

Drug Testing

Diazonium-1H-tetrazole was used as an ‘active-site-directed reagent.’ This substance was designed to interact specifically with amino acid residues, the building blocks of proteins, which are involved in the enzyme’s catalytic reaction:

• In this case, diazonium-1H-tetrazole was found to inactivate the alcohol dehydrogenase enzyme almost stoichiometrically when it reacted with the sulfur atom of a specific cysteine residue, Cys-174.

Model Compound

A ‘model compound’, the tetrazole adduct of free cysteine, was synthesized:

• Elementary and spectral analyses of this adduct showed that the structure was consistent with 5-tetrazoleazo-S-cysteine.
• This compound absorbs light at a maximum wavelength of 316 nm but is destroyed when irradiated at this wavelength.

Enzyme Analysis

The modified, inactive form of the enzyme still bound NADH, a crucial molecule in energy metabolism:

• This was determined through a method called difference spectroscopy.
• However, the modified enzyme did not enhance the fluorescence of the bound NADH, unlike the native enzyme.

Reason for Enzyme Inactivation

X-ray crystallography studies showed that Cys-174 coordinates with zinc at the active site of the enzyme:

• The large, negatively charged tetrazole ring in the modified enzyme likely interferes electrostatically or sterically with the binding of substrates or with hydride transfer.
• Therefore, the modification made to Cys-174 crucially affects the function of the enzyme, thus inactivating it.