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Glycosidases and Glycosyltransferases

Glycosidases and Glycosyltransferases. Introduction to Inverting/Retaining Mechanisms Inhibitor design Chemical Reaction Proposed catalytic mechanisms. Multiple slides courtesy of Harry Gilbert with Wells modifications. Glycosidic bond cleavage. H 2 O. Glycone. Aglycone.

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Glycosidases and Glycosyltransferases

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

  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 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

  18. Glycosylation reactions Glycosyltransferases: Essentials of Glycobiology Chapter 5, Figure 1 Second Edition

  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 • 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

  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 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------------ Kinetics: Initially thought to all be Bi Bi Sequential, Now many appear Random

  25. Strict acceptor substrate specificity of glycosyltransferases Essentials of Glycobiology Chapter 5, Figure 3 Second Edition

  26. Glycan-modifying enzymes Essentials of Glycobiology Chapter 5, Figure 2 Second Edition

  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 412:835-8. (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)

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