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 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?

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

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

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

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

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:

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

Annual Reviews

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

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.)

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.

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

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)