Human Monoglyceride Lipase

EC # 3.1.1.23Jaqueline D. HookerCHE 442: Proteins

April 29, 2010

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

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

2-AGGlycerol

Arachidonic acid

Amino Acid Sequence of Human MGL

• ETGPEDPSSXPEESSPRRTPQSIPYQDLPHLVNADGQYLFCRYWKPTGTPKALIFVSHGAGEHSGRYEELARXLXGLDLLVFAHDHVGHGQSEGERXVVSDFHVFVRDVLQHVDSXQKDYPGLPVFLLGHSXGGAIAILTAAERPGHFAGXVLISPLVLANPESATTFKVLAAKVLNLVLPNLSLGPIDSSVLSRNKTEVDIYNSDPLICRAGLKVCFGIQLLNAVSRVERALPKLTVPFLLLQGSADRLCDSKGAYLLXELAKSQDKTLKIYEGAYHVLHKELPEVTNSVFHEINXWVSQRTATAGTASPP

α/β hydrolaseHomodimer

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

Ser132

His279

Asp249

Catalytic triadBeta sheetsHydrophobic lidAlpha helices

Catalytic Triad

Lid DomainLid Domain

PLVLANPESATTFKVLAAKVLNLVLPNLSLGPIDS

Oxyanion Hole

Ala61

Mse133 (Met133)

Ser132

Asp249

His279

H2O

Inhibition of MGL by Covalent Linkage to Ser132 or Cys Residues

Ser132

Cys252

Cys218

Cys211

Asp249 His279

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

Enzyme Kinetics

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

Table of Important Group and Atom Interactions

Atom 1 Atom 2 DistanceInteraction

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

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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.