Sandbox 50: Difference between revisions
No edit summary |
No edit summary |
||
| Line 24: | Line 24: | ||
===Active Site=== | ===Active Site=== | ||
Trypsin's active site is composed of its catalytic triad, three amino acid residues that are crucial to the enzymes proteolytic function. The catalytic triad consists of Asp 102, His 57, and Ser 195<ref>[Department of Chemistry, University of Maine. The Serine Proteases. http://chemistry.umeche.maine.edu/CHY252/Peptidase3.html]</ref> | Trypsin's active site is composed of its catalytic triad, three amino acid residues that are crucial to the enzymes proteolytic function. The catalytic triad consists of Asp 102, His 57, and Ser 195<ref>[Department of Chemistry, University of Maine. The Serine Proteases. http://chemistry.umeche.maine.edu/CHY252/Peptidase3.html]</ref> | ||
. Serine is the major player in the cleaveage of the peptide bond, thus the name serine protease. His 57 aids in the cleavage of the peptide bond through hydrogen bonds, and Asp 102 aids in the cleavage by electrostatically stabalizing the positively charged form of His 57 in the transition state. Ser 195 performs a nucleophilic attack on the substrate's peptide carbonyl. This causes the oxyanion hole to form. The formation of the oxyanion hole is stabalizing because the carbonyl oxygen that has accepted electrons during the nucleophilic attack fits nicely into this hole, and is stabalized by hydrogen bonds to the backbone NH groups of Gly 193 and Ser 195. A figure of the oxyanion hole can be seen in greater detail in the thumbnail on the left. | . Serine is the major player in the cleaveage of the peptide bond, thus the name serine protease. His 57 aids in the cleavage of the peptide bond through hydrogen bonds, and Asp 102 aids in the cleavage by electrostatically stabalizing the positively charged form of His 57 in the transition state. Ser 195 performs a nucleophilic attack on the substrate's peptide carbonyl. This causes the oxyanion hole to form. The formation of the oxyanion hole is stabalizing because the carbonyl oxygen that has accepted electrons during the nucleophilic attack fits nicely into this hole, and is stabalized by hydrogen bonds to the backbone NH groups of Gly 193 and Ser 195<ref>[Pratt, C.W., Voet, D., Voet, J.G. Fundamentals of Biochemistry - Life at the Molecular Level - Third Edition. Voet, Voet and Pratt, 2008.]</ref> | ||
. A figure of the oxyanion hole can be seen in greater detail in the thumbnail on the left<ref>[Williams, Loren. Georgia Tech. http://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/6521/protein/serine_protease/triad_1.html]</ref>. | |||
The nucleophilic attack by the oxygen of Ser 195 also forms a tetrahedral intermediate. By reconstruction of the carbonyl double bound, the amino portion of the peptide leaves as a product, and an acyl-enzyme intermediate is left in the active site. Now the active site needs to be regenerated. To do this a water molecule nucleophillically attacks the carbonyl carbon, forming another tetrahedral intermediate and reforming the oxyanion hole. The nitrogen of the His 57 ring makes the oxygen of the water more nucleophilic by hydrogen bonding to one of water's hydrogens. By reforming the double bond of the carbonyl carbon, the carboxy end of the original substrate's peptide bond is released, and the active site has been regenerated. The picture in the thumbnail to the left shows the entire catalytic mechanism for a serine protease. | The nucleophilic attack by the oxygen of Ser 195 also forms a tetrahedral intermediate. By reconstruction of the carbonyl double bound, the amino portion of the peptide leaves as a product, and an acyl-enzyme intermediate is left in the active site. Now the active site needs to be regenerated. To do this a water molecule nucleophillically attacks the carbonyl carbon, forming another tetrahedral intermediate and reforming the oxyanion hole. The nitrogen of the His 57 ring makes the oxygen of the water more nucleophilic by hydrogen bonding to one of water's hydrogens. By reforming the double bond of the carbonyl carbon, the carboxy end of the original substrate's peptide bond is released, and the active site has been regenerated. The picture in the thumbnail to the left shows the entire catalytic mechanism for a serine protease. | ||
| Line 30: | Line 31: | ||
==Zymogen: Trypsin Precursor== | ==Zymogen: Trypsin Precursor== | ||
===Trypsinogen=== | ===Trypsinogen=== | ||
A zymogen is an inactive enzyme precursor. Trypsinogen is the zymogen of Trypsin that is secreted by the pancreas and is converted into the active form Trypsin in the duodenum of the small intestine. It is converted into Trypsin by proteolysis. Trypsin needs to be synthesized and secreted in an inactive form to prevent unwanted destruction of other cellular proteins, and also to regulare when and where enzyme activity of Trypsin can occur. | A zymogen is an inactive enzyme precursor. Trypsinogen is the zymogen of Trypsin that is secreted by the pancreas and is converted into the active form Trypsin in the duodenum of the small intestine. It is converted into Trypsin by proteolysis. Trypsin needs to be synthesized and secreted in an inactive form to prevent unwanted destruction of other cellular proteins, and also to regulare when and where enzyme activity of Trypsin can occur<ref>[McDowall, Jennifer. 2010. Trypsin and Chymotrypsin. http://www.ebi.ac.uk/interpro/potm/2003_5/Page1.htm]</ref>. | ||
In order for trypsinogen to be converted to trypsin, a pro-peptide must be cleaved from trypsinogen. The image at the left shows which sequence is removed from trypsinogen, and the active site of trypsin becomes accessible for its protein substrate. | In order for trypsinogen to be converted to trypsin, a pro-peptide must be cleaved from trypsinogen. The image at the left shows which sequence is removed from trypsinogen, and the active site of trypsin becomes accessible for its protein substrate<ref>[Image from: http://virtuallaboratory.colorado.edu/Biofundamentals/lectureNotes/AllGraphics/trypsinogen.gif]</ref>. | ||
The rate of conversion of trypsinogen to trypsin can be enhanced with calcium ions. Both trypsinogen and trypsin contain an autolysis loop. This autolysis loop is comprised of residues 143-151, and contains a high affinity calcium ion binding site that is required for stability. When the calcium ion is not present, autolysis (self-digestion) will occur. | The rate of conversion of trypsinogen to trypsin can be enhanced with calcium ions. Both trypsinogen and trypsin contain an autolysis loop. This autolysis loop is comprised of residues 143-151, and contains a high affinity calcium ion binding site that is required for stability. When the calcium ion is not present, autolysis (self-digestion) will occur<ref>[Trypsin. 2010. 30 Oct. 2010. http://www.worthington-biochem.com/try/default.html]</ref> | ||
. | |||
[[Image:Trypsinogen.gif |thumb]] | [[Image:Trypsinogen.gif |thumb]] | ||
==Trypsin Inhibition== | ==Trypsin Inhibition== | ||
Trypsin inhibitors are generally known as serine protease inhibitors or serpins. Serpins act as competitive inhibitors so they bind to the trypsin active site, rendering the enzyme inactive. There are four natural sources of trypsin inhibitors -- bovine pancreas, ovomucoid, soybeans, and lima beans. Each of these natural sources of inhibition work in different ways. Inhibitors from soybeans and lima beans inactivate insect proteases, acting as a feeding deterrent. Inhibitors from soybeans have also been found to cause pancreatic hypertrophy in rats, also acting as a feeding deterrent in this case. Other trypsin inhibitors include Ag+, benzamidine, ethylenediaminetetraacetic acid EDTA, and diisopropylfluorophosphate (DFP). | Trypsin inhibitors are generally known as serine protease inhibitors or serpins. Serpins act as competitive inhibitors so they bind to the trypsin active site, rendering the enzyme inactive. There are four natural sources of trypsin inhibitors -- bovine pancreas, ovomucoid, soybeans, and lima beans. Each of these natural sources of inhibition work in different ways. Inhibitors from soybeans and lima beans inactivate insect proteases, acting as a feeding deterrent. Inhibitors from soybeans have also been found to cause pancreatic hypertrophy in rats, also acting as a feeding deterrent in this case. Other trypsin inhibitors include Ag+, benzamidine, ethylenediaminetetraacetic acid EDTA, and diisopropylfluorophosphate (DFP)<ref>[Sigma-Aldrich. 2010. Trypsin Inhibitors. http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/trypsin/trypsin-inhibitors.html]</ref>. | ||
===When Control Fails=== | ===When Control Fails=== | ||
When the balance between trypsin and its inhibitors, or of the activation of trypsin from trypsinogen is disturbed, pancreatitis can result. When trypsinogen is converted to trypsin early (i.e. in the pancrease), it causes the autodigestion of the pancreas. Those with cystic fibrosis often suffer from pancreatitis because it is thought that cystic fibrosis interferes with the negative feedback loop that regulates pancreatic trypsinogen secretions. When there is a high concentration of trypsin in the duodenum, a signal is sent back to the pancrease to reduce the production of trypsinogen. A high concentration of inhibitors induces pancreatic trypsinogen secretion. | When the balance between trypsin and its inhibitors, or of the activation of trypsin from trypsinogen is disturbed, pancreatitis can result. When trypsinogen is converted to trypsin early (i.e. in the pancrease), it causes the autodigestion of the pancreas. Those with cystic fibrosis often suffer from pancreatitis because it is thought that cystic fibrosis interferes with the negative feedback loop that regulates pancreatic trypsinogen secretions. When there is a high concentration of trypsin in the duodenum, a signal is sent back to the pancrease to reduce the production of trypsinogen. A high concentration of inhibitors induces pancreatic trypsinogen secretion<ref>[McDowall, Jennifer. 2010. Trypsin and Chymotrypsin. http://www.ebi.ac.uk/interpro/potm/2003_5/Page1.htm]</ref> | ||
. | |||
| Line 47: | Line 50: | ||
<ref>[Gorga, F. (2007, March 12). Disulfide bonds. http://webhost.bridgew.edu/fgorga/proteins/disulfide.htm]</ref> | <ref>[Gorga, F. (2007, March 12). Disulfide bonds. http://webhost.bridgew.edu/fgorga/proteins/disulfide.htm]</ref> | ||
<ref>[Department of Chemistry, University of Maine. The Serine Proteases. http://chemistry.umeche.maine.edu/CHY252/Peptidase3.html]</ref> | <ref>[Department of Chemistry, University of Maine. The Serine Proteases. http://chemistry.umeche.maine.edu/CHY252/Peptidase3.html]</ref> | ||
<ref>[]</ref> | <ref>[Pratt, C.W., Voet, D., Voet, J.G. Fundamentals of Biochemistry - Life at the Molecular Level - Third Edition. Voet, Voet and Pratt, 2008.]</ref> | ||
<ref>[Williams, Loren. Georgia Tech. http://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/6521/protein/serine_protease/triad_1.html]</ref> | |||
<ref>[McDowall, Jennifer. 2010. Trypsin and Chymotrypsin. http://www.ebi.ac.uk/interpro/potm/2003_5/Page1.htm]</ref> | |||
<ref>[Image from: http://virtuallaboratory.colorado.edu/Biofundamentals/lectureNotes/AllGraphics/trypsinogen.gif]</ref> | |||
<ref>[Sigma-Aldrich. 2010. Trypsin Inhibitors. http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/trypsin/trypsin-inhibitors.html]</ref> | |||