1. Human Monoglyceride Lipase EC # 3.1.1.23 Jaqueline D. Hooker CHE 442: Proteins April 29, 2010

2. Introduction Endocannabinoids (neuromodulatory lipids), such as 2- arachidonoylglycerol (2-AG) and N-arachidonoyl- ethanolamide (AEA), act as signaling molecules They are produced by neurons and released “on demand” They are efficiently catabolized to ensure rapid signal inactivation Monoglyceride lipase (MGL), a serine hydrolase, catalyzes the hydrolysis of 2-AG to arachidonic acid and glycerol Inhibitors can irreversibly, covalently bind to Ser132 Cysteine-reactive agents act as inhibitors of MGL – Steric hindrance of active site entrance – Steric hindrance of critical Ser residue

3. Human Monoglyceride Lipase α/β hydrolase Is a homodimer of module A and module B – Module A is used as a representative structure of the protein – Does not require both modules to function 2-methyl-pentane-2,4-diol (MPD) was present in the active site for successful crystallization (not shown) 4 MPD molecules in A and 3 molecules in B – The additional molecule of MPD is thought to stabilize the lid domain of A

4. Reaction Mechanism for MGL 2-AG Glycerol Arachidonic acid

5. Amino Acid Sequence of Human MGL ETGPEDPSSXPEESSPRRTPQ SIPYQDLPHLVNADGQYLFCR YWKPTGTPKALIFVSHGAGE HSGRYEELARXLXGLDLLVF AHDHVGHGQSEGERXVVSD FHVFVRDVLQHVDSXQKDYP GLPVFLLGHSXGGAIAILTAA ERPGHFAGXVLISPLVLANPE SATTFKVLAAKVLNLVLPNLS LGPIDSSVLSRNKTEVDIYNS DPLICRAGLKVCFGIQLLNAV SRVERALPKLTVPFLLLQGSA DRLCDSKGAYLLXELAKSQD KTLKIYEGAYHVLHKELPEVT NSVFHEINXWVSQRTATAGT ASPP α/β hydrolase Homodimer

6. Multiple Sequence Alignment Amino Acids part of Catalytic Triad Amino Acids part of the Oxyanion Hole Amino Acids critical for Inhibition Amino Acids with Hydrophobic Interactions with Inhibitors *The amino acid sequence is well conserved; the amino acids important for interaction are highlighted

7. MGL Active Site of Module A Ser132 His279 Asp249 Catalytic triad Beta sheets Hydrophobic lid Alpha helices Catalytic Triad

8. Lid Domain PLVLANPESATTFKVLA AKVLNLVLPNLSLGPIDS

9. Oxyanion Hole Ala61 Mse133 (Met133) Ser132 Asp249 His279 H2OH2O

10. Inhibition of MGL by Covalent Linkage to Ser132 or Cys Residues Ser132 Cys252 Cys218 Cys211 Asp249 His279

11. One Molecule with Inhibition Ability Irreversibly binds (covalently) to MGL – Ser132 Modifies catalytic Ser residue Prevents catalysis because it is a non- hydrolyzable substrate – Cys252 Sterically hinders Ser132 Blocks active site, prevents interaction with catalytic Ser132 N-arachidonylmaleimide (NAM) Resembles 2-AG

12. Enzyme Kinetics

13. Substrate Stabilization of the Polar Head of 2-AG Lys170 Glu164 Asn162 Ser132 His279 Asp249 Lid Domain Interact with Polar head group *stabilize via dipole interactions Catalytic amino acids

14. Table of Important Group and Atom Interactions Atom 1Atom 2Distance Interaction Type Ser132 OHis279 N2.61 ÅIonic Ser132 OAsp249 O6.94 ÅIonic Ala61 NHOH O2.90 ÅIonic Mse133 NHOH O3.15 ÅIonic Cys252 SHis279 N2.39 Å Hydrogen Bond Cys252 SAsp249 O13.29 Å Hydrogen Bond Cys252 SAsp249 O23.27 Å Hydrogen Bond

15. Conclusions Catalytic triad (Ser, His, Asp) is required for function Oxyanion hole is used to stabilize carbonyl carbon of the tetrahedral intermediate Site close to catalytic triad for glycerol to diffuse from the active site – Prevents association of glycerol with hydrophobic lid domain Active site lid recruits lipid molecules due to hydrophobic/neutral nature of the amino acids Inhibition occurs by – Blocking the active site by covalently binding to Cys residue – Covalent bond to critical Ser residue Therefore, we now possess a better understanding of the regulation of [2-AG] by MGL

16. References Bertrand, T. et al. (2010). J. Mol. Biol., 396, 663- 673. Labar, G. et al. (2010). ChemBioChem, 11, 218-227. Karlsson, M. et al. (1997). J. Biol. Chem., 272, 27218-27223 Saario, S.M. and Laitinen, J.T. (2007). Chemistry & Biodiversity, 4, 1903-1913. Savinainen, J.R., et al.(2010). Analytical Biochemistry. 309: 132-134. Senior, J.R. and Isselbacher, K.J. (1963). Journal of Clinical Investigation, 42, 187-195.