lecture020309 - Primary objectives of these lectures...

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Primary objectives of these lectures Discuss how enzymes catalyze chemical reactions My examples will be Glycosyl hydrolases Lysozyme Glycosyl transferases Describe how chemical reactions are speeded up Discuss experimental evidence for 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 are in same plane So positive charge is not just at C1 but shared between C1 and ring oxygen This stabilises positive charge. Need lots of energy to cause change in geometry of sugar O C1 O5
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Two different mechanisms of acid-base assisted catalysis Single displacement 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 Double displacement mechanism Retention of the anomeric configuration of glycone sugar β-glycosidic bond Bond is equatorial β –glycone sugar: 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 Glutamate or aspartate Two mechanisms are as follows:
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Acid-base assisted single displacement 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-base assisted double displacement mechanism Two distinct reactions Glycosylation Formation of a covalent glycosyl-enzyme intermediate (ester bond) The aglycone sugar released from active site Deglycosylation The ester bond between the glycone sugar and the enzyme is hydrolysed and the glycone sugar is released from the active site 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
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This note was uploaded on 03/23/2009 for the course BCMB 8130 taught by Professor Wells,tiemeyer during the Spring '09 term at University of Georgia Athens.

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lecture020309 - Primary objectives of these lectures...

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