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lecture020309 - Primary objectives of these lectures...

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Primary objectives of theselectures Discuss how enzymes catalyzechemical reactions My examples will be Glycosyl hydrolases Lysozyme Glycosyl transferases Describehow chemical reactions arespeeded up Discuss experimental evidencefor proposed catalytic mechanisms
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Glycosidic bond cleavage O O O O OH OH O Example is lysozyme Discovered by Alexander Fleming in 1920s Something fell from his nose onto his bacterial agar plate Bacteria lysed Potential antimicrobial enzyme He discovered a better antimicrobial agent later; what is it? Glycone Aglycone H 2 O
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Glycosidic bond cleavage in free solution Glycone Aglycone H 2 O O O O O OH OH O O HO HO OH O OH O HO OH OH OH H + O O HO HO OH OH OH OH - O HO HO OH OH OH + Transition state oxocarbenium ion attacked by hydroxyl ion
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Rate of glycosidic bond cleavage The transition state (positively charged oxocarbenium ion) is a very high energy molecule Geometry changes from chair to half-chair Why? So C1 and ring oxygen arein same plane So positivechargeis not just at C1 but shared between C1 and ring oxygen This stabilises positivecharge. Need lots of energy to causechange in geometry of sugar O C1 O5
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Two different mechanisms of acid-base assisted catalysis Singledisplacement mechanism Inversion of the anomeric configuration of glycone sugar β-glycosidic bond Bond is equatorial α -glycone sugar OH is axial
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Two different mechanisms of acid-base assisted catalysis Doubledisplacement mechanism Retention of theanomeric configuration of glycone sugar β-glycosidic bond Bond is equatorial β –glyconesugar: OH remains equatorial
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Two different mechanisms of acid-base assisted catalysis How does an enzyme generate protons and hydroxyl ions? Two amino acids with carboxylic acid side-chains Glutamateor aspartate Two mechanisms are as follows:
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Acid-base assisted singledisplacement mechanism The acid catalyst Uncharged Hydrogen in the perfect position to be donated to the glycosidic oxygen . The catalytic base Extracts a proton from water Hydroxyl ion in the perfect position to attack C1 of the transition state Catalytic base Catalytic acid
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Acid-baseassisted double displacement mechanism Two distinct reactions Glycosylation Formation of a covalent glycosyl-enzymeintermediate(ester bond) Theaglyconesugar released fromactivesite Deglycosylation Theester bond between theglyconesugar and theenzymeis hydrolysed and theglyconesugar is released fromthe activesite Catalytic acid-base Catalytic nucleophile
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Hen egg white lysozyme Hen egg white lysozyme The first enzyme structure solved The textbook example of enzyme catalyzed glycoside hydrolysis Hydrolyses the glycosidic bond via a retaining mechanism
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Three possible mechanisms for lysozyme Three possible mechanisms for lysozyme O R'O R''O OH NH O R O O R'O
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