04 dallas Lipase complete2 1

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					Lipase




         Biocomputing Project
         Presented By: Angel, Jason,
                       Jeff and Kelly
            Outline
• Background Information
• Structure
• Phylogeny/Homology
              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
   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
    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
  Background Information
• Pancreatic Colipase (PC)
  – Secreted as procolipase in a 1:1 ratio with PL
  – PC can bind to the bile salt micelle
  – PC necessary for PL function
Structure of Lipase
Overview
• Has only one protein chain (Chain A)
  – 534 amino acids long
  – 12 β sheets
  – 25 ά helices
• No DNA chains
• Has two Disulfide bridges
• Has six ligands
Ligands
1. (1R)-Menthyl hexyl phosphonate group
                          •Center of the protein
                          •Connected to lots of
                          different Amino Acids
                          •Two Serines and two
                          leucines
2. – 3. Calcium Ions

              2. Calcium Ion A
               •Interacts with
               Asparagine, amino
               acid # 260
               •Also with one
               hetero atom
2. – 3. Calcium Ions

             3. Calcium Ion B
              •Interacts with
              Glycine, amino acid #
              326
              •Connected to
              multiple heteroatoms
 4. N-Acetyl-D-Glucosamine (NAG)

•Interacts with asparagine
222, lysine 180,
asparagine 314, Valine
313, and tryptophan 221.
 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
Active Site
• Not referenced in Protein Explorer, so
  determined via conserved regions.
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).
Structural Motifs
• Multiple phosphorylation sites
• Contains much N-terminal glycine which
  have a covalent bond with myristate (a
  C-14 saturated fatty acid).
 Predictive Software Analysis
                        Ά Helices Β Sheets

           Predicted        11           11

             Actual         25           12

Generally predicted the locations of helices and
sheets, but not with perfect accuracy.
 Transmembrane Properties
 • Lipase is not an integral membrane
   protein




•No interesting relatives
                  Phylogeny
Homologs to 1LPM ‘lipase’
•   Gram negative bacteria
•   Homo sapien carboxylesterase
•   Possum carboxylesterase
•   Domestic horse butyrylcholinesterase
•   Homo sapien acetylcholinesterase
•   Roundworm acetylcholinesterase
 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
Multiple Sequence Alignment

                        •More conserved
                        residues


                        •Not many
                        conserved groups
Phylogenetic Tree

• Unrooted



Values used to
determine
bootstrapping values
Phylogenetic Tree



•Calculated
Bootstrapping
                    100%
values
                           100%



                100%              88.4%
What's interesting?
• Expected bootstrapping values
  closer to 100%
• Actually 88.4%
• Less Confidence
                       REFERENCES
• Svendsen A (2000). "Lipase protein engineering". Biochim
  Biophys Acta 1543 (2): 223–228.
• Winkler FK, D'Arcy A, and W Hunziker (1990). "Structure of
  human pancreatic lipase". Nature 343 (6260): 771–774.

                    Algorithms Used
• Protein Explorer
   – Jmol
   – Consurf
   – Biology Workbench (PELE, TMAP, GREASE, TMHMM,
     BLASTP, PROSEARCH, CLUSTALW, CLUSTALTREE,
      DRAWTREE ).

				
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