lec 13 - Glycosidase cleaves polysaccharides at glycosidic...

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Glycosidase cleaves polysaccharides at glycosidic bonds between sugars. Enzyme uses strategy of lowering AE, by distorting the substrate into the conformation which resembles the transition state. Six sugars that bound to enzyme, 5 out of 6 negative delta G, but binding of sugar to the side D large positive energy. Otherwise, one looks at total binding energy, (-RTlnK), if we look at equilibrium constant of binding. Add free energies and multiply individual constants. Good binding, making use of negative energies. The mechanism is associated with catalytic steps involves two carboxylic side chains: free acid form of GLU 35 (proton donor) and other carboxylic acid side chain is in its conjugate base form. And transition state of catalytic cleavage state we make acid hydrolyze the glycocidic linkage and resulting product of sugar and side D, is cation the structure is accommodated by keeping ring in ½ chair confirmation. This stabilizes the transition state. The fact that there are 6 binding sites, the substrate bound to the enzyme before catalysis has net favorable free energy. Enzyme substrate complex has lower free energy then free enzyme and free substrate. BUT transition state is stabilized even more, bc of half chair confirmation sugar. When enzyme binds substrate and there is no catalysis--- transition state is dropped even more when it is associated with enzyme, stabilizing enzyme product or enzyme substrate. But transition is the most stabilized. The covalent intermediate is feature of many other enz catalyzed reaction, same two amino acids. But when glu was acting as acid and asp as base. The different situation is vice versa--- glu as base and asp as acid. End of hydrolysis of covalent intermediate convert back to base as asp and acid as glu. Active side of ribonuclease: the hydrolysis of alternating polymer of phosphoric acid and ribose. When amino acid side chain finds charged environment (positive charges) acid give off proton very easily and conjugate base is formed when it is buried in hydrophobic env---holds on to its proton. Amino acids that participate in hydrolysis of ribose phosphate polymeric backbone are two HISTIDINES , distinctive NMR spectrums of Histidines. We know which protons have dissociated and which have not. The
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lec 13 - Glycosidase cleaves polysaccharides at glycosidic...

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