Alcohol desydrogenase (ADH) catalyses the interconversion of ethanol and other alcohols with their corresponding aldehydes and ketones. ADH is one of the best known enzymes in terms of molecular structure. The active site of ADH contains a ZN atom and remains accessible only through a hydrophobic tunnel. The amino acid residues near the ZN atom and those surrounding the hydrophobic tunnel confer peculiar substrate specificities to different ADH forms. Human ADH Class III/ formaldehyde deshydrogenase displays a high substrate specificity towards omega-hydroxyacids and its natural substrate S-hydroxymethylglutathione. It has been postulated that Arg 304 is properly positioned for interaction with the carbonyl groups from these substrates. The aim of the project proposal is to study the structure-function relationships in ADH using ADH Class III as a model. The following steps will be undertaken 1) Expression of human liver ADH (ClassIII) in E. coli.2) Purification and characterization of recombinant ADH ClassIII.3) Computerized molecular modelling of the tridimensional structure of ADH Class III, based on the crystallographic coordinates of horse ADH.4) Side-directed mutagenesis of the residues of the hydrophobic tunnel. 5) Side-directed mutagenensis of Arg 304 6) Side-directed mutagenesis of amino acid residues surrounding the catalytic ZN 7) purification and characterization of mutant proteins
|Effective start/end date||1/04/91 → 31/03/92|
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