1. Glycosidases and Glycosyltransferases
Introduction to Inverting/Retaining Mechanisms
Inhibitor design
Chemical Reaction
Proposed catalytic mechanisms
Multiple slides courtesy of Harry Gilbert with Wells modifications

2. Glycosidic bond cleavage
H2O
Glycone
Aglycone
Classic example is lysozyme: cleaves N-acetlymuramic acid-b-4-GlcNAc
Discovered by Alexander Fleming in 1920s
Sneezed onto his bacterial agar plate
Bacteria found to be lysed next day
Potential antimicrobial enzyme
He discovered a better antimicrobial agent later; what is it?

3. Glycosidic bond cleavage in free solution
Glycone
Aglycone
H2O
Transition state oxocarbenium
ion attacked by hydroxyl ion

4. 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 O5 C1

5. 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
sugar
OH is axial

6. 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
OH remains equatorial

7. 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:

8. Acid-base assisted single displacement mechanism
Catalytic acid
Catalytic base
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

9. Acid-base assisted double displacement mechanism
Catalytic acid-base
Catalytic nucleophile
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

10. 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
Exceptions to the Rules Exist

11. And the lysozyme mechanism is revisited: Covalent enzyme intermediate for hen egg white lysozyme
Lysozyme (E35Q)
Asp52
Vocadlo et al. Nature 412, 835-8

12. Inhibitors of glycoside hydrolases
Glycoside hydrolase activities contribute to significant diseases
Flu
Type II diabetes
Possibly Cancer and Aids
To combat diseases need to develop inhibitors

13. Designing glycoside hydrolase inhibitors
What comprises a good inhibitor?
Mechanistic covalent inhibitors not used
Very high affinity non-covalent competitive inhibitors
Transition state inhibitors

14. The retaining mechanism
glycosylation
Transition state has a
positive charged nature as leaving group departure
precedes nucleophile attack
deglycosylation

15. TS-based inhibitors that mimic charge distribution
deoxynojirimycin
Both have nM Ki values.
Affinities are about one
million times higher
than substrate
Glucosidase Inhibitors
isofagamine
Why are they transition state mimics?
Contains a positive charge

16. Mimicking the half-chair
Insert a double-bond to enforce planarity

17. Drugs that mainly mimic the half chair All picomolar affinities 108-fold tighter binders than substrates
HIV drug: prevents glycosylation in mammalian cells
AIDs virus surface proteins are not glycosylated
and thus can’t evade the immune system
Type II diabetes
(inhibits human
Amylase)
Anti-flu
drugs

18. Glycosylation reactions
Glycosyltransferases:
Chapter 5, Figure 1
Glycosylation reactions
Essentials of Glycobiology
Second Edition
FIGURE 5.1. Glycosylation reactions. A glycosyltransferase uses a glycosyl donor and an acceptor substrate. In animals, glycosyl donors include nucleotide sugars and dolichol-phosphate-linked monosaccharides and oligosaccharides. Bacteria also use undecaprenyl-pyrophosphate (PP)-linked donors. Acceptors are most commonly oligosaccharides, but (in rare cases) they can be monosaccharides. Proteins and ceramides are also acceptors for the glycosyltransferases that initiate glycoprotein, proteoglycan, and glycolipid synthesis. Many other targets, such as drugs and other small molecules, can be glycosylated, but these are not discussed in this chapter. Even DNA can be glycosylated.

19. Two folds
Both have two Rossman domains
GTA strongly linked may look like a single b-sheet—originally thought to all have D-X-D and metal at active site
GT-B has two separate domains—originally thought to be metal independent
Requirement of nucleotide binding appears to limit number of folds greatly

20. Inverting GT
Retaining GT
Currently being disputed
Why?
No Donor-Enz intermediate
can be found
SNi mechanism?
Substrate-assisted catalysis?

21. How can we identify the catalytic amino acids
Glycoside hydrolases and transferases are grouped in enzyme families based on sequence similarity (i.e. evolved from a common ancestor. Currently 100+ families
All members of same family have
Evolved from the same progenitor sequence
Conserved mechanism
Same fold
Conserved catalytic apparatus

22. CAZY (for hydrolases and transferases)
Several families have ancient ancestral relationship
Same fold, mechanism and catalytic residues
How does CAZY help us?
Tells us what the catalytic residues are
Tells us the mechanism
Tells us the likely substrate specificity

23. Annual Reviews

24. Catalytic acid
Sequence : QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN
1 UNIPROT:XYNA_PSEFL 1:73 335: QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN
2 UNIPROT:Q9AJR :68 111: RHNQQVRGHNLCWHE--ELPTwaSEVngNAKEILIQHIQTVAGRYAGRIQSWDVVNEAILPKDGRPDG-----
3 UNIPROT:GUX_CELFI 3:66 115: GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFADG-DGP
4 UNIPROT:Q :61 116: GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFAD
5 UNIPROT:Q :63 324: ENNMTVHGHALVWHSDYQVPnwAGSAE-DFLAALDTHITTIVDHYegNLVSWDVVNEAIDDNS
6 UNIPROT:Q :63 343: NNINVHGHALVWHSDYQVPNFmsGSAADFIAEVEDHVTQVVTHFkgNVVSWDVVNEAINDGS
7 UNIPROT:Q :73 111: QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMAHYKGKIVQWDVVNEAFADG--NSGGRRDSN
8 UNIPROT:Q7SI :73 73: QNGKQVRGHTLAWHS--QQPGWMQssGSTLRQAMIDHINGVMGHYKGKIAQWDVVNEAFSD--DGSGGRRDSN
9 UNIPROT:XYNB_THENE 1:62 96: KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS
10 UNIPROT:Q :62 96: KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS
11 UNIPROT:AAN :62 96: KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS
12 UNIPROT:Q7TM : : GHTVVWHGA--VPTWLNasTDDFRAAFENHIRTVADHFRGKVLAWDVVNEAV---ADDGSG-----
13 UNIPROT:Q7WVV :62 96: ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS
14 UNIPROT:Q7WUM :62 96: ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS
15 UNIPROT:Q9WXS :62 96: ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS
16 UNIPROT:Q9P :57 120: QNGKSIRGHTLIWHS--QLPAWVNnnNAdlRQVIRTHVSTVVGRYKGKIRAWDVVNE
17 UNIPROT:Q9X :63 115: QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIAQWDVVNEAFADGS
18 UNIPROT:XYNA_STRLI 1:63 114: QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS
19 UNIPROT:Q8CJQ :63 114: QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS
20 UNIPROT:P :62 93: QNGQGLRCHTLIWYS--QLPGWVSSGNWN-RQTLEahIDNVMGHYKGQCYAWDVVNEAVDDN
21 UNIPROT:Q9XDV :71 427: GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS--
22 UNIPROT:Q8GJ :71 427: GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS--
23 UNIPROT:Q7X2C : :88 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMNHSKGKIAQWDVVNEAFADGS
24 UNIPROT:Q9RJ :61 105: GMDVRGHTLVWHS--QLPSWVSPLGadLRTAMNAHINGLMGHYKGEIHSWDVVNEAFQD
25 UNIPROT:Q :61 119: GMKVRGHTLVWHS--QLPGWVSPLAadLRSAMNNHITQVMTHYKGKIHSWDVVNEAFQD
26 UNIPROT:Q9RMM :61 113: QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED
27 UNIPROT:BAD :61 113: QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED
Kinetics: Initially thought to all be Bi Bi Sequential, Now many appear Random

25. Strict acceptor substrate specificity of glycosyltransferases
Chapter 5, Figure 3
Essentials of Glycobiology
Second Edition
FIGURE 5.3. Strict acceptor substrate specificity of glycosyltransferases as exemplified by the human B blood group α1-3 galactosyltransferase. The B transferase adds galactose in α1-3 linkage to the H antigen (top). This enzyme requires the α1-2-linked fucose modification of the H antigen for activity because the B transferase does not add to an unmodified type-2 precursor (middle), or precursors modified by sialyl residues (bottom) or other monosaccharides (not shown). (For the monosaccharide symbol code, see Figure 1.5, which is reproduced on the inside front cover.)

26. Glycan-modifying enzymes
Chapter 5, Figure 2
Essentials of Glycobiology
Second Edition
FIGURE 5.2. Glycan-modifying enzymes. A variety of donors are used to modify glycans.

27. Take Home Points CAZY Inverting/Retaining Mechanisms Specificity
Mechanistic Based Inhibitors

28. References Cantarel et al (2008) Nucleic Acid Res 37:D233-8 (CAZY)
Vocadlo at al. (2001) Nature 412: (Mechanistic inhibitors of glycoside hydrolases)
Lairson et al. (2008) Ann. Rev. Biochem. 77: (glycosyltransferases)
Rye and Withers (2000) Curr. Opin. Chem. Biol. 4: (glycoside hydrolases)
Tailford (2008) Nature Chem. Biol. Nat. 4: (Transition state geometry)