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Human Monoglyceride Lipase EC # 3.1.1.23 Jaqueline D. Hooker CHE 442: Proteins April 29, 2010 Introduction • Endocannabinoids (neuromodulatory lipids), such as 2arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (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 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 Reaction Mechanism for MGL Arachidonic acid 2-AG Glycerol 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 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 MGL Active Site of Module A Catalytic Triad His279 Ser132 Asp249 Catalytic triad Beta sheets Hydrophobic lid Alpha helices Lid Domain Lid Domain PLVLANPESATTFKVLA AKVLNLVLPNLSLGPIDS Oxyanion Hole Mse133 (Met133) Ser132 Asp249 H2O His279 Ala61 Inhibition of MGL by Covalent Linkage to Ser132 or Cys Residues Cys218 Cys252 Cys211 Ser132 Asp249 His279 One Molecule with Inhibition Ability • Irreversibly binds (covalently) to MGL – Ser132 • Modifies catalytic Ser residue • Prevents catalysis because it is a nonhydrolyzable substrate – Cys252 N-arachidonylmaleimide (NAM) Resembles 2-AG • Sterically hinders Ser132 • Blocks active site, prevents interaction with catalytic Ser132 Enzyme Kinetics Substrate Stabilization of the Polar Head of 2-AG Lid Domain Interact with Polar head group *stabilize via dipole interactions Catalytic amino acids Asn162 Ser132 His279 Asp249 Lys170 Glu164 Table of Important Group and Atom Interactions Atom 1 Atom 2 Distance Interaction Type Ser132 O His279 N 2.61 Å Ionic Ser132 O Asp249 O 6.94 Å Ionic Ala61 N HOH O 2.90 Å Ionic Mse133 N HOH O 3.15 Å Ionic Cys252 S His279 N 2.39 Å Hydrogen Bond Cys252 S Asp249 O1 3.29 Å Hydrogen Bond Cys252 S Asp249 O2 3.27 Å Hydrogen Bond 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 References Bertrand, T. et al. (2010). J. Mol. Biol., 396, 663673. 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.