Glycosyl

Transcription

Now let’s study the mechanism of how these glycosidase enzymes carry out their chemistry. These are the enzymes we use to determine α or β for our disaccharide, lactose. And the glycosidase that we’re going to study is lysozyme which is a naturally occurring antibiotic, it’s found for example in human tears. It causes bacteria cell walls to weaken and undergo- and the cell to- to ah, lyse. What is does is it cleaves specifically at the sugar GlcNAc, which is the sugar shown here which has a 2 amino group as the acetamides. So, the acetamide position at C2 rather than having a hydroxyl group. That specific glycoside bond is cleaved, it’s a β-glycosidase that cleaves this β-glycoside bond. But, the key point that I want you to take away from the slide is that it does so with retention of stereochemistry at that anomeric position. This bl- β- glycoside bond is cleaved and the hydroxyl group on the resulting product, muramic acid is this β hydroxyl group. So, how does it cleave with retention of stereochemistry? Some of the details of the mechanisms are still under debate, but they key point and- and idea on how this retention of stereochemistry comes about is shown here. In the active side are two carboxylic acid side chains at position 35, is the neutral ah, form of the ah, side chain [psk] glutamate, glutamic acid. And at position 52 is another carboxylic acid from aspartate, it’s in its negatively charged form and it can serve as a nucleophile. Well, the way this reaction is believed to take place to hydrolyze this bond here is to use that glutamic acid as a general acid that can ah, protonate that leaving hydroxyl groups. So, protonation of this oxygen atom produces the hydroxyl group at that C4 position- the C4 position of muramic acid. [psk] Now, the ah, oxocarbenium ion intermediate - that’s a word that you should be familiar with and you might want to write down – this oxocarbenium ion intermediate is a very potent electrophile. And the nearby nucleophile which is on the α-face, the underside, will undergo attack and stabilize as a covalent adduct through that aspartate oxygen. The- in- in a α linkage the ah, oxocarbenium ion is trapped in that way. What happens next at the ah, position is formation of oxocarbenium ion again. So, if we go back to the same intermediate, but it’s held in this form and in fact this has been isolated, this c- covalent adduct has been isolated. But, it will revert back to the oxocarbenium ion and then in the presence of water, we have the general base, the glutamic acid now as its glutamate at position 35, deprotonates the water which adds into the α-face. And so, this whole point of the aspartate 52 is really to block the α-face. It’s the β-face that’s left open that when water enters into the active site, that’s the only site- that’s the only face that’s available for reaction. And so, the hydroxyl group enters, we have a β-anomeric configuration that’s pre- preserved. Basically, what you see is we’ve done a double inversion. That is, here you can see the stereochemistry was inverted, but then in the final product since that α-face was blocked by this nucleophilic aspartate group entering only the β-face, we’ve basically done a double inversion. And that result is retention of configuration.