Lipase

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Lipase
Biocomputing Project Presented By Angel, Jason,
Jeff and Kelly
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Outline

• Background Information
• Structure
• Phylogeny/Homology

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Overview

• Lipase responsible for fat absorption in the
  small intestine
• Its activity depends on presence of pancreatic
  colipase and bile salts
• Lipase insufficiency can be managed by diet
• Lipase malabsorption can be induced by Xenical ?
  weight loss

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Background Information

• Lipases hydrolyze triacylglycerols, releasing one
  fatty acid at a time and forming diacylglycerols,
  monoacylglycerol then finally, glycerol.
• Lipase is essential in most if not all living
  organism
• Lipases also used by fungi and bacteria to
  facilitate nutrient absorption from the external
  medium

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Background Information

• Pancreatic Lipase
• Released into duodonem
• Hydrolyzes triacylglycerols (TAG) into free fatty
  acids and monoglycerides
• TAGs are emulsified within bile salt micelles

Main enzyme responsible for fat absorption in
small intestine
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Background Information

• Pancreatic Colipase (PC)
• Secreted as procolipase in a 11 ratio with PL
• PC can bind to the bile salt micelle
• PC necessary for PL function

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Structure of Lipase
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Overview

• Has only one protein chain (Chain A)
• 534 amino acids long
• 12 ß sheets
• 25 ? helices
• No DNA chains
• Has two Disulfide bridges
• Has six ligands

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Ligands
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1. (1R)-Menthyl hexyl phosphonate group

• Center of the protein
• Connected to lots of different Amino Acids
• Two Serines and two leucines

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2. 3. Calcium Ions
2. Calcium Ion A

• Interacts with Asparagine, amino acid 260
• Also with one hetero atom

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2. 3. Calcium Ions
3. Calcium Ion B

• Interacts with Glycine, amino acid 326
• Connected to multiple heteroatoms

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4. N-Acetyl-D-Glucosamine (NAG)

• Interacts with asparagine 222, lysine 180,
  asparagine 314, Valine 313, and tryptophan 221.

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5. NAG group

• Two NAG ligands interacting with each other at
  the same site.
• Also interact with Tyrosine 299, aparagine 351,
  valine 352, glutamine 357, and glutamate 70

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Active Site

• Not referenced in Protein Explorer, so determined
  via conserved regions.

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Protein Family

• Multiple possibilities
• Carboxylesterase (E of 6e-91)
• Lipolytic Enzyme (E of 2.8e-5)
• Hydrolase (E of 0.19)
• However, often classified as a Hydrolase by
  various databanks (such as GenBANK file in NCBI).

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Structural Motifs

• Multiple phosphorylation sites
• Contains much N-terminal glycine which have a
  covalent bond with myristate (a C-14 saturated
  fatty acid).

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Predictive Software Analysis
Generally predicted the locations of helices and
sheets, but not with perfect accuracy.
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Transmembrane Properties

• Lipase is not an integral membrane protein

• No interesting relatives

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Homologs to 1LPM lipase
Phylogeny

• Gram negative bacteria
• Homo sapien carboxylesterase
• Possum carboxylesterase
• Domestic horse butyrylcholinesterase
• Homo sapien acetylcholinesterase
• Roundworm acetylcholinesterase

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E-Values
E-values of Homologs
e-value Monodelphis domestica 7e-41
Xanthomonas 8e-43 Homo sapien
acetlycholinesterase 7e-40 Homo sapien
carboxylesterase 4e-35 Meloidogyne
1e-41 Equus caballus 1e-41
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Multiple Sequence Alignment

• More conserved residues
• Not many
• conserved groups

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Phylogenetic Tree

• Unrooted

Values used to determine bootstrapping values
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Phylogenetic Tree

• Calculated
• Bootstrapping
• values

100
100
100
88.4
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What's interesting?

• Expected bootstrapping values closer to 100
• Actually 88.4
• Less Confidence

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REFERENCES

• Svendsen A (2000). "Lipase protein engineering".
  Biochim Biophys Acta 1543 (2) 223228.
• Winkler FK, D'Arcy A, and W Hunziker (1990).
  "Structure of human pancreatic lipase". Nature
  343 (6260) 771774.
• Algorithms Used
• Protein Explorer
• Jmol
• Consurf
• Biology Workbench (PELE, TMAP, GREASE, TMHMM,
  BLASTP, PROSEARCH, CLUSTALW, CLUSTALTREE,
  DRAWTREE ).