1. Primary objectives of these lectures
Discuss how enzymes catalyze chemical reactions
My examples will be
Glycosyl hydrolases
Lysozyme
Glycosyl transferases
Describe how chemical reactions are speeded up
Discuss experimental evidence for proposed catalytic mechanisms

2. Glycosidic bond cleavage
H2O
Glycone
Aglycone
Example is lysozyme
Discovered by Alexander Fleming in 1920s
Something fell from his nose onto his bacterial agar plate
Bacteria lysed
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
α-glycone 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
β –glycone sugar:
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

11. Three possible mechanisms for lysozyme
Covalent glycosyl-enzyme
Asp 52
Ion-pair with long-lived
glycosyl cation
Covalent oxazoline
(anchimeric assistance)

12. Accumulation of a 2F-chitobiosyl lysozyme (E35Q) intermediate
14315 Da
GlcNAc2FGlcF
Lysozyme (E35Q)
14864 Da
Lysozyme (E35Q)
14316 Da
Mass (Da)
2F-Chitobiosyl-lysozyme (E35Q)
Free lysozyme

13. Stable covalent intermediate
Only the high molecular weight molecule observed
Suggests the formation of a covalent substrate-enzyme intermediate
How do we prove this?
Solve the 3D structure of this protein
Will show if there is a covalent bond between the substrate and enzyme

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

15. How can we identify the catalytic amino acids
Glycoside hydrolases 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

16. CAZY 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

17. 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

19. Why did the active centre of mannosidases look like that of glucosidases ?

20. Covalent Intermediate
OS2 skew-boat
Catalytic base
Catalytic base
Proves the identity of the catalytic nucleophile
and shows sugar distortion
Why is this remotely interesting?

21. Strong support for B2,5 transition-state
Enzymes perform mannoside chemistry by placing O2 pseudo-equatorial
The “difference” between mannosidases and glucosidases is actually seen at O3

22. 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

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

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

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

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

27. 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

28. Flu
RNA virus
Uses membrane of its host into which it inserts its own proteins
Major prevention of disease?
Vaccination
Pandemic
Killed 20 million
New pandemic about to occur. Bird flu coming from Far East China
Estimated will die in UK as a result of the new strain

29. Nucleotide-donor
domain
Acceptor domain
Open conformation
closed conformation
Structure and function of glycosyltransferases that glycosylate macrolide
antibiotics: Underpins the modulation of the specificity and potency of these
antibiotics

30. Annual Reviews

31. Two folds
Both have two Rossman domains
GTA strongly linked may look like a single b-sheet
GT-B has two separate domains
Requirement of nucleotide binding limits number of folds greatly

32. Inverting GT
Retaining GT

33. Annual Reviews

34. Inverting GT
Retaining GT

35. Annual Reviews

37. 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)