Human Monoglyceride Lipase EC # 3.1.1.23

1. Human Monoglyceride LipaseEC # 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 Arachidonic acid 2-AG Glycerol

5. Amino Acid Sequence of Human MGL • etgpedpssxpeessprrtpqsipyqdlphlvnadgqylfcrywkptgtpkalifvshgagehsgryeelarxlxgldllvfahdhvghgqsegerxvvsdfhvfvrdvlqhvdsxqkdypglpvfllghsxggaiailtaaerpghfagxvlisplvlanpesattfkvlaakvlnlvlpnlslgpidssvlsrnktevdiynsdplicraglkvcfgiqllnavsrveralpkltvpflllqgsadrlcdskgayllxelaksqdktlkiyegayhvlhkelpevtnsvfheinxwvsqrtatagtaspp 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 Catalytic Triad His279 Ser132 Asp249 Catalytic triad Beta sheets Hydrophobic lid Alpha helices

8. Lid Domain Lid Domain plvlanpesattfkvlaakvlnlvlpnlslgpids

9. Oxyanion Hole Mse133 (Met133) Ser132 Asp249 H2O Ala61 His279

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

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 Lid Domain Interact with Polar head group *stabilize via dipole interactions Catalytic amino acids Asn162 Lys170 Ser132 Glu164 His279 Asp249

14. Table of Important Group and Atom Interactions

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.