Lysozyme Flashcards
What is lysozyme an example of?
Strain.
What does lysozyme catalyse?
Degradation of bacterial cell walls by hydrolysing the β-1,4-glycosidic bonds between NAM and NAG sugar units.
Where does the polysaccharide bind within lysozyme?
In a groove, with binding sites for 6 residues, where Asp52 and Glu35 react to break the scissile bond.
What is the length dependancy of the polysaccharide on the kcat of lysozyme? How was it investigated?
Need at least 5 residues for an efficient turnover number. Investigated using different length polysaccharide substrate analogues.
How can the length dependancy be explained?
D binding site has an unfavourable binding energy and catalysis occurs between sites D and E, meaning there must be sugars bound in sites A, B, C, E and F for there to be an overall favourable ΔG.
Why does the D binding site have a lower binding energy?
The sugar in the D site adopts the half-chair conformation which is more energetically unfavourable than the chair conformation and experiences more steric strain- when binding to the D site.
What is the role of Glu35 in the Philips mechanism for lysozyme?
Acts as a general acid and protonates the oxygen in the glycosidic bond.
What happens once the glycosidic bond has been cleaved in the Philips mechanism?
Leaves a positively charged D-ring oxonium ion.
How is the D-ring oxonium ion stabilised in the Philips mechanism?
By favourable electrostatic interaction with the negatively charged carboxylate of Asp52 and enzyme-induced distortion of the D-ring to enhance resonance structure stabilisation.
What is the role of water in the Philips mechanism?
Water from the solution provides an OH that combines with the oxonium ion and the proton from the water is used to reprotonate Glu35.
What types of catalysis are involved in the Philips mechanism?
Acid-base catalysis and electrostatic catalysis.
Why can the D-ring oxonium ion in the Philips mechanism only be stabilised when in the half-chair conformation?
Needs the p-orbital overlap in the half chair conformation to stabilise the delocalisation of the positive charge.
Name the two possible mechanisms for the lysozyme reaction.
Philips mechanism and the Koshland mechanism.
What type of catalysis is involved in the Koshland mechanism?
Covalent catalysis.
What is the role of Asp52 in the Koshland mechanism?
Acts as a nucleophile and attacks at the anomeric carbon in an Sn2 type reaction.
