A. B. C. D. E. F. G. H. I. J. K. Ser/Thr. Ser/Thr. Ser/Thr. Ser/Thr. Ser/Thr. Asn. Asn. Asn. Asn. Asn. Asn

Transcription

1 A. B. C. D. E. F. "3 "3!4!3 Ser/Thr "3!4!3!4 Asn Asn Ser/Thr Asn!3!6 Ser/Thr G. H. I. J. K.!3 Ser/Thr Ser/Thr Asn Asn Asn

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

3 Glycosidic bond cleavage Glycone Aglycone H 2 H H Classic example is lysozyme: cleaves N-acetlymuramic acid-β-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?

4 Glycosidic bond cleavage in free solution Glycone Aglycone H 2 H H + H H H H H H H H H Transition state oxocarbenium ion attacked by hydroxyl ion + H H H H H - H H H H H H

5 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 5 C1

6 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 H is axial

7 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 H remains equatorial

8 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:

9 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

10 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

11 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

12 And the lysozyme mechanism is revisited: Covalent enzyme intermediate for hen egg white lysozyme H H H AcHN H H F Lysozyme HEWL (E35Q) relative intensity (E) m/z (E-I) min min 50 min 33 min 17 min 150 min 120 min 90 min Asp52 intensity Relative Intensity Vocadlo et al. Nature 412, Da 15000

13 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

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

15 The retaining mechanism H H H H R H H H δ - δ + R δ + H δ - glycosylation Transition state has a positive charged nature as leaving group departure precedes nucleophile attack H H H H H H H H H H H δ - δ + H H H δ + H δ - deglycosylation

16 TS-based inhibitors that mimic charge distribution deoxynojirimycin H Glucosidase Inhibitors isofagamine H H H H NH 2 Both have nm K i values. Affinities are about one million times higher than substrate Why are they transition state mimics? H H NH 2 + Contains a positive charge

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

18 Drugs that mainly mimic the half chair All picomolar affinities 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) H H H H CH 3 HN H H H Acarbose Miglitol H H H H H H N H H H H H H H H AcNH AcNH HN NH NH 2 NH 2 C 2 H Relenza C 2 H Tamiflu Anti-flu drugs

19 Glycosylation reactions Essentials of Glycobiology Second Edition Chapter 5, Figure 1

20 Two folds Both have two Rossman domains GTA strongly linked may look like a single β- sheet GT-B has two separate domains Requirement of nucleotide binding appears to limit number of folds greatly

21 Inverting GT Retaining GT

22 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

23 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

24 Annual Reviews

25 Catalytic acid Sequence 1:73 QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN 1 UNIPRT:XYNA_PSEFL 1:73 335:407 QNGQTVHGHALVWHPSYQLPNWASDSNANFRQDFARHIDTVAAHFAGQVKSWDVVNEALFDSADDPDGRGSAN 2 UNIPRT:Q9AJR9 1:68 111:178 RHNQQVRGHNLCWHE--ELPTwaSEVngNAKEILIQHIQTVAGRYAGRIQSWDVVNEAILPKDGRPDG UNIPRT:GUX_CELFI 3:66 115:176 --GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFADG-DGP UNIPRT:Q :61 116:173 --GKELYGHTLVWHS--QLPDWAKNLNGsfESAMVNHVTKVADHFEGKVASWDVVNEAFAD UNIPRT:Q :63 324:391 ENNMTVHGHALVWHSDYQVPnwAGSAE-DFLAALDTHITTIVDHYegNLVSWDVVNEAIDDNS UNIPRT:Q :63 343:409 -NNINVHGHALVWHSDYQVPNFmsGSAADFIAEVEDHVTQVVTHFkgNVVSWDVVNEAINDGS UNIPRT:Q :73 111:180 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMAHYKGKIVQWDVVNEAFADG--NSGGRRDSN 8 UNIPRT:Q7SI98 1:73 73:142 QNGKQVRGHTLAWHS--QQPGWMQssGSTLRQAMIDHINGVMGHYKGKIAQWDVVNEAFSD--DGSGGRRDSN 9 UNIPRT:XYNB_THENE 1:62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS UNIPRT:Q :62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS UNIPRT:AAN :62 96:158 KNDMIVHGHTLVWHN--QLPGWLTgsKEELLNILEDHVKTVVSHFRGRVKIWDVVNEAVSDS UNIPRT:Q7TM36 8:68 2: GHTVVWHGA--VPTWLNasTDDFRAAFENHIRTVADHFRGKVLAWDVVNEAV---ADDGSG UNIPRT:Q7WVV0 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS UNIPRT:Q7WUM6 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS UNIPRT:Q9WXS5 1:62 96:158 ENDMIVHGHTLVWHN--QLPGWITgtKEELLNVLEDHIKTVVSHFKGRVKIWDVVNEAVSDS UNIPRT:Q9P973 1:57 120:176 QNGKSIRGHTLIWHS--QLPAWVNnnNAdlRQVIRTHVSTVVGRYKGKIRAWDVVNE UNIPRT:Q9X584 1:63 115:176 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIAQWDVVNEAFADGS UNIPRT:XYNA_STRLI 1:63 114:175 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS UNIPRT:Q8CJQ1 1:63 114:175 QNGKQVRGHTLAWHS--QQPGWMQssGSALRQAMIDHINGVMAHYKGKIVQWDVVNEAFADGS UNIPRT:P :62 93:155 QNGQGLRCHTLIWYS--QLPGWVSSGNWN-RQTLEahIDNVMGHYKGQCYAWDVVNEAVDDN UNIPRT:Q9XDV5 3:71 427:505 --GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS-- 22 UNIPRT:Q8GJ37 3:71 427:505 --GMKVHGHTLVWHQ--QTPAWMndSGGNirEemRNHIRTVIEHFGDKVISWDVVNEAMSDNPSNpdWRGS-- 23 UNIPRT:Q7X2C9 1:63 27:88 QNGKQVRGHTLAWHS--QQPGWMQssGSSLRQAMIDHINGVMNHSKGKIAQWDVVNEAFADGS UNIPRT:Q9RJ91 3:61 105:162 --GMDVRGHTLVWHS--QLPSWVSPLGadLRTAMNAHINGLMGHYKGEIHSWDVVNEAFQD UNIPRT:Q :61 119:176 --GMKVRGHTLVWHS--QLPGWVSPLAadLRSAMNNHITQVMTHYKGKIHSWDVVNEAFQD UNIPRT:Q9RMM5 1:61 113:172 QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED UNIPRT:BAD :61 113:172 QNGKEVRGHTLAWHS--QQPYWMQssGSDLRQAMIDHINGVMNHYKGKIAQWDVVNEAFED

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

27 Take Home Points CAZY Inverting/Retaining Mechanisms 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. pin. Chem. Biol. 4: (glycoside hydrolases) Tailford (2008) Nature Chem. Biol. Nat. 4: (Transition state geometry)