Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.788581
Title: Substrates and inhibitors of diamine oxidase
Author: Barr, Stephen David
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Abstract:
This thesis describes a study of the enzyme pea seedling diamine oxidase (ps DAO) and concentrates on four main areas: (1) enzyme-catalysed oxidation of primary diamines by pea seedling diamine oxidase (2) oxidation of pyridine-amines by pea seedling diamine oxidase (3) inhibition of diamine oxidase and (4) further purification of pea seedling diamine oxidase. (1) Oxidation of Primary Diamines by Pea Seedling Diamine Oxidase: Pea seedling diamine oxidase catalyses the oxidation of a range of diamines into the corresponding aminoaldehydes according to scheme A. Putrescine (i) and cadaverine (ii) are the natural substrates for the enzyme. Although much work has been carried out to determine the relative effectiveness of these compounds and analogues thereof as substrates for pea seedling diamine oxidase, little data existed concerning the substrate potential of cyclic diamines. A range of simple substituted aromatic and alicyclic diamines were synthesised in the form of the dihydrochloride salts and tested as substrates for the enzyme using an improved spectrophotometric assay system. The assay is dependent on the accurate measurement of hydrogen peroxide, produced as a product during the enzymatic oxidation. From the assay KM and Vmax values were obtained directly, giving an indication of the relative binding affinities and rate of oxidation of the various substrates. Analysis of these results suggested that although the cyclic diamines were poorer substrates than the natural substrates they did in general show a greater binding affinity for the enzyme than either putrescine (i) or cadaverine (ii). Comparison of the kinetic parameters obtained for a range of substituted aromatic diamines provided information on the effect of the position and nature of the substituents in determining the binding affinity and rate of oxidation of these compounds. Comparison between the kinetic parameters found for analogues of the natural substrate with those of the natural substrate itself often provides useful clues into the nature of the active site. A range of putrescine analogues were tested as substrates for pea seedling diamine oxidase. All attempts to synthesise optically active 2-haloputrescines (iii) met with failure. A range of 2,3-dihaloputrescines (iv) were however synthesised as the dihydrochlorides and kinetic data was collected for these substrates. (2) Oxidation of Pyridine-Amines by Pea Seedling Diamine Oxidase: Pea seedling diamine oxidase is capable of catalysing the oxidation of some primary monoamines, albeit at reduced rates compared to the corresponding diamines. In order to examine more fully the role played by the second amine group during catalysis a range of (aminoalkyl)pyridine derivatives (v) and (vi) were synthesised as the corresponding dihydrochloride salts and assayed as substrates for the enzyme. The results obtained suggest that the presence of a second primary amine group is not required for efficient substrate binding however it does have a more definite role to play in promoting the deamination. (3) Inhibition of Pea Seedling Diamine Oxidase: The oxidative deamination of diamines by diamine oxidase is a key step in polyamine metabolism. Polyamines are known to be essential for cell growth and replication. Inhibitors of the reaction catalysed by diamine oxidase may have a detrimental effect on polyamine metabolism and therefore on cell growth. A range of compounds shown by our initial kinetic studies to be poor substrates but efficient binders for the enzyme were tested as inhibitors of pea seedling diamine oxidase. The tests were carried out using a modified version of the spectrophotometric assay used previously. Many of the compounds were shown to inhibit competitively the diamine oxidase catalysed deamination of putrescine. Ki values are reported for all compounds tested. (4) Further Purification of Pea Seedling Diamine Oxidase: Pea seedling diamine oxidase can readily be isolated in a partially purified form from peas by means of a series of precipitations. The enzyme prepared in this manner was suitable for our kinetic studies. In an attempt to produce a homogeneous enzyme preparation a method was developed which utilised, as the final purification step, the technique of Fast Protein Liquid Chromatography. Although a homogeneous preparation was not obtained the 280-fold increase in specific activity observed compares well with previous purifications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.788581  DOI: Not available
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