Primary objectives of these lectures

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 • http://afmb.cnrs-mrs.fr/CAZY/ • 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 1:73 QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN 1 UNIPROT:XYNA_PSEFL 1:73 335:407 QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN 2 UNIPROT:Q9AJR9 1:68 111:178 RHNQQVRGHNLCWHE--ELPTwaSEVngNAKEILIQHIQTVAGRYAGRIQSWDVVNEAILPKDGRPDG----- 3 UNIPROT:GUX_CELFI 3:66 115:176 --GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFADG-DGP------- 4 UNIPROT:Q59277 3:61 116:173 --GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFAD------------ 5 UNIPROT:Q59675 1:63 324:391 ENNMTVHGHALVWHSDYQVPnwAGSAE-DFLAALDTHITTIVDHYegNLVSWDVVNEAIDDNS---------- 6 UNIPROT:Q59301 2:63 343:409 -NNINVHGHALVWHSDYQVPNFmsGSAADFIAEVEDHVTQVVTHFkgNVVSWDVVNEAINDGS---------- 7 UNIPROT:Q59139 1:73 111:180 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMAHYKGKIVQWDVVNEAFADG--NSGGRRDSN 8 UNIPROT:Q7SI98 1:73 73:142 QNGKQVRGHTLAWHS--QQPGWMQssGSTLRQAMIDHINGVMGHYKGKIAQWDVVNEAFSD--DGSGGRRDSN 9 UNIPROT:XYNB_THENE 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS----------- 10 UNIPROT:Q60044 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS----------- 11 UNIPROT:AAN16480 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS----------- 12 UNIPROT:Q7TM36 8:68 2:58 -------GHTVVWHGA--VPTWLNasTDDFRAAFENHIRTVADHFRGKVLAWDVVNEAV---ADDGSG----- 13 UNIPROT:Q7WVV0 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS----------- 14 UNIPROT:Q7WUM6 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS----------- 15 UNIPROT:Q9WXS5 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS----------- 16 UNIPROT:Q9P973 1:57 120:176 QNGKSIRGHTLIWHS--QLPAWVNnnNAdlRQVIRTHVSTVVGRYKGKIRAWDVVNE---------------- 17 UNIPROT:Q9X584 1:63 115:176 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIAQWDVVNEAFADGS---------- 18 UNIPROT:XYNA_STRLI 1:63 114:175 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS---------- 19 UNIPROT:Q8CJQ1 1:63 114:175 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS---------- 20 UNIPROT:P79046 1:62 93:155 QNGQGLRCHTLIWYS--QLPGWVSSGNWN-RQTLEahIDNVMGHYKGQCYAWDVVNEAVDDN----------- 21 UNIPROT:Q9XDV5 3:71 427:505 --GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS-- 22 UNIPROT:Q8GJ37 3:71 427:505 --GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS-- 23 UNIPROT:Q7X2C9 1:63 27:88 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMNHSKGKIAQWDVVNEAFADGS---------- 24 UNIPROT:Q9RJ91 3:61 105:162 --GMDVRGHTLVWHS--QLPSWVSPLGadLRTAMNAHINGLMGHYKGEIHSWDVVNEAFQD------------ 25 UNIPROT:Q59922 3:61 119:176 --GMKVRGHTLVWHS--QLPGWVSPLAadLRSAMNNHITQVMTHYKGKIHSWDVVNEAFQD------------ 26 UNIPROT:Q9RMM5 1:61 113:172 QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED------------ 27 UNIPROT:BAD02382 1:61 113:172 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. glycosylation deglycosylation The retaining mechanism Transition state has a positive charged nature as leaving group departure precedes nucleophile attack

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 chairAll 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 1918-199 • New pandemic about to occur. Bird flu coming from Far East China • Estimated 50000 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 412:835-8. (Mechanistic inhibitors of glycoside hydrolases) • Lairson et al. (2008) Ann. Rev. Biochem. 77:521-555 (glycosyltransferases) • Rye and Withers (2000) Curr. Opin. Chem. Biol. 4:573-580 (glycoside hydrolases) • Tailford (2008) Nature Chem. Biol. Nat. 4:306-12 (Transition state geometry)