Download Human Complement C3 Protein

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
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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.