lectures 48-51 - glycosyltransferases and glycosylhydrolases

Question 1

what do we call the bond that holds two sugars together?

what is type of bond is this?

Answer 1

• glycosidic bond

- actually an acetal bond

Question 2

what are the two ways in which we characterize glycosidic bonds? provide an example

Answer 2

(1) relative stereochemistry of the anomeric carbon
- if the C is equitorial = beta, if it is axial = alpha
(2) linkage number
- what carbon does the bond exist btwn?
- e.g. if the bond is btwn C1 and C3 = B 1,3 linkage

Question 3

what is the anomeric carbon

Answer 3

• the carbon of the acetal of hemiacetal bond

- call this “C1”

Question 4

what are glycosyltransferases (GTs)?

Answer 4

• enzymes that catalyze the formation of glycoside bonds to form glycosdies (glycans)
• i.e. elongate sugar chains

Question 5

what are glycosylhydrolases (GHs)? (aka glycosides)

Answer 5

• enzymes that catalyze the hydrolysis of glycosidic bonds

- i.e. shorten sugar chains

Question 6

describe (generally) the mechanism of action for glycosyltransferases

Answer 6

• the sugar that contains the anomeric carbon is the glycosyl donor - gives its sugar
• anomeric carbon is the electrophile and has a leaving group
• the sugar that does not have the anomeric carbon is the glycosyl acceptor
• OH on its sugar acts as the nucelophile

Question 7

the glycosyl donor for GTs is typically what?

Answer 7

• typically a nucleotide sugar, where the nucleotide acts as the LG
• can also be a lipid (e.g. dilichol phosphate)

Question 8

describe UDP-gal as a glycosyl donor

Answer 8

• composed of galactose sugar bound to a diphosphate, bound to a ribose sugar, bound to uridine
• galactose sugar is what is being donated
• leaving group = UDP
• diphosphate makes it a good leaving group

Question 9

what are other nucleotide sugars involved in GTs

Answer 9

UDP-GlcNac, UDP-GalNac, GDP-Man, GDP-Fuc, CMP-NeuSac (sialic acid)

Question 10

do GTs have a high or low degree of specificity for their donor and acceptor substrates? what does this contribute to?

Answer 10

• high

- many GTs are namd based on the monosaccaride they transfer to the growing sugar chain

Question 11

GTs catalyse reactions with one of two effects on sterochemistry, what are the effects?

Answer 11

inversion or retention

Question 12

have there been proven cases of retention in GTs? if so provide an example

Answer 12

no, only GHs

Question 13

what does inversion refer to specifically? what sort of mechanism if it?

Answer 13

• stereochemistry of the glycosyl donor
• starts with the one linkage, then the stereochemistry is inverted to the resulting glycoside
• these typically follow SN2 like mechanisms of nucelophilic displacement

Question 14

provide a general example for the mechanism of inversion

Answer 14

• a residue on the GT that acts as an acid interacts with the glycosyl donor to neutralize the charge created when the C-O bond btwn the anomeric carbon and the LG breaks
• alternatively, instead of an acid, can have a DXD motif (where X is any aa) with a metal ion bound that also acts to stabilize the neg charge of the LG
• similarly, a residue acting as a base on the GT interacts with the glycosyl acceptor to facilitate the removal of an h+ from the hydroxyl group, making the oxygen a better nucleophile, allowing the SN2 like displacement of the LG
• inversion happens where the initial sugar was an alpha linkage and the product is a beta linkage

Question 15

what are requirements in the active site for a beta 1,4 galactosyltransferases

Answer 15

• DVD motif at 252,253,254
• D residue at 318
• divalent ions (e.g. MG2+)

Question 16

describe the role of the active site requirements for beta 1,4 galactosyltransferases

Answer 16

• D res at 318 (top) acts as the base to deprotonate the c4 OH group of the acceptor
• O can then act as a Nu(-) and attack the donor
• D’s of DVD coordinate the Mg2+ and help position it to the diphosphates
• Mg2+ helps with the charge production after Nu(-) attack to stabilize phosphates and facilitate cleavage of the LG

Question 17

where do GHs cleave the glycosidic bond? what is the result

Answer 17

• btwn the anomeric carbon and the O (of the glycosylic bond)
• first sugar gains the OH from the water
• result = two sugars

Question 18

is inverting of GHs similar to inverting of GTs? what is the difference?

Answer 18

• you betchya, still switch from alpha to beta or vice versa

- the only difference is now the nucleophile comes from water (not another sugar)

Question 19

describe the retaining actions of GHs

Answer 19

• basically a double SN2-like displacement reaction separated by a glycosyl-enzyme intermediate
• instead of res that acts like a base in inverting rxns, have a res that acts as a nucleophile and protonates the C-O bond linked to the LG, giving the LG a h+ to facilitate its cleavage
• still have acid at the bottom
• result is a covalent glycosyl-enzyme complex where the Nu is now covalently bonded to the sugar where the LG was
• the acid at the bottom now acts as a base, grabbing a h+ from a water, facilitating the second SN2 rxn - the addition of an OH grp to the sugar (where second sugar used to be)
• final product is a sugar with the same stereochemistry

Question 20

provide an example of a retaining GHs

Answer 20

• sialidases (aka neuraminidases)

- cleave sialic acids (aka neuraminic acids) resulting in products with the same stereochemistry

Question 21

how do we denote the stereochemistry for sialic acids?

Answer 21

we denote stereochemistry based on the carboxylic acid not the anomeric carbon

Question 22

describe the key elements of influenza neuroiminidase and the function of these key elements (3)

Answer 22

(1) 2 glu residues (one acts to deprotonate tyr, then reprotonate it, other acts to protonate the LG then deprotonate h2o to be the 2nd nucleophile)
(2) tyr acts as the first nucleophile
(3) arg residues (not involved in catalysis) forms a salt bridge to stabilize the carboxylate group of sialic acid

Question 23

describe the mechanism of action for neuroiminidase

Answer 23

• top deprotonated glu res deprotonates tyr to make it more nucleophilic
• tyr acts as the nucleophile and cleaves the glycosidic bond
• bottom protonated glu res protonates the LG to facilitate it leaving
• result is 1st SN2 reaction - resulting in the covalent glycosyl enzyme complex
• the now deprotonated bottom glu deprotonates a water to make it a nucelophile
• OH attacks the sugar
• now protonated top glu protonates the tyr, cleaving the covalent bond btwn tyr and the sugar
• the result is the second SN2 reaction, producing the final sugar with retention of its origional stereochemistry

Question 24

why are neuroimidases important?

Answer 24

pharmeceutical target for the influenze virus

Question 25

the microbiological term for influenza is H1N1, what do the H and the N refer to?

Answer 25

proteins found on the viral envolope:

• H = hemagglutinin (HA)
• N = neuraminidase (Neu)

Question 26

what is the role of HA proteins in the progression of influenza?

Answer 26

• HAs are responsible for the initial attachement of flu virus capsids to host cell surfaces so they can be endocytosed
• recognize and bind sialyated GPs on host cell membranes (need sialic acid present to be endocytosed)

Question 27

what is the role of Neu proteins in the progression of influenza?

Answer 27

• involved in virus budding

- cleave sialic acid to allow the release of new virus particles from host cells

Question 28

how do Neu inhibitors function?

Answer 28

bind Neu receptors and prevent the release of new virus capsids, reducing pathogenicity of the virus

Question 29

describe the structure of Neu inhibitors

Answer 29

these are mimetics of sialic acid (have similar structures)