Enzymatic studies of alcohol dehydrogenase by a combination of in vitro and in silico methods

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Sammanfattning: The family of alcohol dehydrogenases (ADHs) catalyzes conversions of alcohols, ketons and aldehydes. The early discovery and isolation of ADH (1937) was followed by numerous investigations. It was also the first dimeric enzyme for which the threedimensional structure was determined (1974). Recent findings have revealed new physiological functions for the ADH enzymes. One type is a key enzyme in hepatic retinol metabolism, another is a main formaldehyde scavenger and a regulator of Snitrosothiols levels. ADH genes have been shown to be connected to diseases and syndromes, such as alcoholism and asthma. Hence, investigations of structure-function relationships of the ADH enzymes are of both physiological and medical interest. The aim of this thesis was to study structure-function relationships and to investigate and identify ligands for ADH, with in vitro and in silico methods. The catalytic activities of human, mouse and rat ADH2 for retinoids, were determined. The Km values for human ADH2 are the lowest among all known human dehydrogenases, which supports a key role for human ADH2 in the hepatic retinoid metabolism. ADH3 is an enzyme with a proposed role as an NO scavenger. Two new lines of ligands, bile acids and fatty acids, were investigated for their potential effects on NO homeostasis. The bronco dilatatory effect of NO suggests that ADH3 inhibition could potentially work as treatment of obstructive lung disorders. The stability of the quaternary structure of sorbitol dehydrogenase (SDH) was determined by in vitro experiments and in silico energy calculations. A hydrogen-bonding network crucial for the tetrameric stability in SDH was identified. This network is located at a region enclosing the structural zinc site in mammalian ADHs. The structural zinc site was studied in detail by a combination of molecular dynamics and quantum mechanics simulations. The simulations revealed that the interaction between the cysteine residues and the zinc atom is of an electrostatic and covalent nature. With in silico and in vitro simulations, interactions between ligands and the active site were determined, revealing site specific interactions within both ADH2 and ADH3. Furthermore, studies of subunit interactions and the structural zinc site revealed properties of the quaternary stability.

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