Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.344679
Title: A study of the arom enzyme complex by limited proteolysis
Author: Smith, David Drummond Stewart
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1980
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Abstract:
An improved method of purifying the arom complex from N. crassa has been devised. This procedure takes advantage of the tight binding of the arom complex to blue-dextran sepharose and yields around 3mg of pure enzyme from 100g of lyophilised N. crassa mycelia (30-35%). Limited proteolysis has been used to probe the domain structure of the arom complex. The shikimate kinase activity of the complex is very susceptible to inactivation by proteolysis and is probably located on the surface of the arom complex. A 69000 molecular weight domain containing both the shikimate dehydrogenase and dehydroquinase activities of the arom complex has been characterised by a combination of gel electrophoresis in the presence of IM urea, 8M urea and SDS and by substrate-labelling of the dehydroqmnase active-site. The shikimate dehydrogenase-dehydroquinase proteolytic fragment of the arom complex can regain shikimate dehydrogenase activity after polyacrylamide gel electrophoresis in the presence of 8M urea. This domain of the arom complex can refold in the absence of the remainder of the arom polypeptide. Under the same conditions shikimate dehydrogenase activity was not regained from the arom complex after polyacrylamide gel electrophoresis in the presence of 8M urea. Evidence has been obtained that a proteolytic fragment of molecular weight 110000 is produced from the N-terminal region of the arom polypeptide and may catalyse both the dehydroqmnate synthetase and EPSP synthetase reactions of the complex. Chemical modification of the arom complex with formaldehyde/ sodium borohydride inactivated both the shikimate dehydrogenase and dehydroquinase activities of the complex. Shikimate protected the shikimate dehydrogenase but not the dehydroquinase activity against inactivation by reductive alkylation. This and other evidence indicated that the shikimate dehydrogenase and dehydroquinase active-sites are spatially distinct.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.344679  DOI: Not available
Keywords: Biochemistry
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