Catalytic Strategies
13.3 هزار بار بازدید -
14 سال پیش
-
This course is part of
This course is part of a series taught by Kevin Ahern at Oregon State University on General Biochemistry. For more information about online courses go to http://ecampus.oregonstate.edu/ Playlist
1. Proteases catalyze the hydrolysis of peptide bonds in polypeptides. They are usually fairly specific for certain amino acids and cut at or near those amino acids.
2. Chymotrypsin is a protease whose activity has been closely studied. Conveniently, the activity of chymotrypsin can be studied using an artificial substrate which, when cleaved by the enzyme, releases a yellow product.
3. When the release of the colored substrate by the enzyme is studied, it appears to occur in two different rates. First there is a VERY rapid release of the colored substrate. After that initial burst of activity, the remaining yellow color is released slowly.
4. The reason appears to be that the reaction catalyzed occurs in two steps. The first step cleaves the bond to produce the yellow product. The product of this release is that the remainder of the substrate is covalently linked to the enzyme. In order for the enzyme to bind another substrate molecule and release more yellow color, it must first release the covalently bound molecule. This step occurs slowly and explains why subsequent yellow molecules are released slowly - after the initial one is released, the enzyme must remove the covalently bound molecule, bind a new substrate, and cut the substrate and the continue the process repeatedly.
5. Chymotrypsin is an example of a protease that employs reactive serine in its active site. Such an enzyme is called a serine protease. Treatment of chymotrypsin with DIPF, which covalently links to serines, inactivates the enzyme.
6. Serine proteases form covalent intermediates with their polypeptide substrates. The first step involves nucleophilic attack of an alkoxide ion on the polypeptide substrate to form an acyl-enzyme intermediate. Formation of this intermediate results in cleavage of the peptide bond and release of the first polypeptide fragment. The acyl-enzyme intermediate is resolved by addition of water to release the other portion of the original polypeptide along with regeneration of the original enzyme active site. This last step occurs relatively slowly.
7. In the active site of chymotrypsin (and other serine proteases) is a so-called catalytic triad of amino acids that includes a serine hydrogen bonded to a histidine. The histidine is, in turn, hydrogen bonded to an aspartic acid residue in the active site. Each of the hydrogen bonds of the catalytic triad is important in the catalytic mechanism. The nucleophilic alkoxide ion on serine is made possible ultimately by interactions in the catalytic triad and these hydrogen bonds.
8. The catalytic triad is not unique to chymotrypsin. The serine protease known as subtilisin also has the same catalytic triad and employs a similar mechanism.
9. Besides the catalytic triad, the enzyme has two other important sites to consider. The first is the oxyanion hole that stabilizes a tetrahedral intermediate that arises during the catalysis. The second, called as S1 pocket, is where the substrate binds. Both the oxyanion hole and the S1 pocket are adjacent to the active site (catalytic triad).
10. The S1 pocket determines a serine protease's specificity. The S1 pocket of chymotrypsin is hydrophobic and relatively large, allowing it to bind phenylalanine, for example. Remember that chymotrypsin cuts adjacent to phenylalanine (among other hydrophobic amino acids). The S1 pocket of trypsin, for example has a negatively charged group in the bottom, allowing it to bind to lysine or arginine.
11. Other proteases include cysteine proteases (use cysteine and histidine in the active site), aspartyl proteases (use aspartic acids and water in the active site) metalloproteases (use a metal ion - usually zinc - and water in the active site).
11. Carbonic anhydrase is an enzyme that catalyzes the joining of carbon dioxide and water to form carbonic acid.
12. A zinc ion (held in place by three histidines in the active site of carbonic anhydrase) plays an important role in the catalysis of the enzyme by binding a water molecule. A subsequent loss of a proton by water is necessary for catalysis. Notably, the enzyme has maximal activity at a high pH (where protons are easily removed) and a lower activity in an acidic pH (6.0).
13. The limiting step in the action of carbonic anhydrase is the abstraction of the proton from water. Buffers and/or bases help facilitate this and thus speed the reaction.
1. Proteases catalyze the hydrolysis of peptide bonds in polypeptides. They are usually fairly specific for certain amino acids and cut at or near those amino acids.
2. Chymotrypsin is a protease whose activity has been closely studied. Conveniently, the activity of chymotrypsin can be studied using an artificial substrate which, when cleaved by the enzyme, releases a yellow product.
3. When the release of the colored substrate by the enzyme is studied, it appears to occur in two different rates. First there is a VERY rapid release of the colored substrate. After that initial burst of activity, the remaining yellow color is released slowly.
4. The reason appears to be that the reaction catalyzed occurs in two steps. The first step cleaves the bond to produce the yellow product. The product of this release is that the remainder of the substrate is covalently linked to the enzyme. In order for the enzyme to bind another substrate molecule and release more yellow color, it must first release the covalently bound molecule. This step occurs slowly and explains why subsequent yellow molecules are released slowly - after the initial one is released, the enzyme must remove the covalently bound molecule, bind a new substrate, and cut the substrate and the continue the process repeatedly.
5. Chymotrypsin is an example of a protease that employs reactive serine in its active site. Such an enzyme is called a serine protease. Treatment of chymotrypsin with DIPF, which covalently links to serines, inactivates the enzyme.
6. Serine proteases form covalent intermediates with their polypeptide substrates. The first step involves nucleophilic attack of an alkoxide ion on the polypeptide substrate to form an acyl-enzyme intermediate. Formation of this intermediate results in cleavage of the peptide bond and release of the first polypeptide fragment. The acyl-enzyme intermediate is resolved by addition of water to release the other portion of the original polypeptide along with regeneration of the original enzyme active site. This last step occurs relatively slowly.
7. In the active site of chymotrypsin (and other serine proteases) is a so-called catalytic triad of amino acids that includes a serine hydrogen bonded to a histidine. The histidine is, in turn, hydrogen bonded to an aspartic acid residue in the active site. Each of the hydrogen bonds of the catalytic triad is important in the catalytic mechanism. The nucleophilic alkoxide ion on serine is made possible ultimately by interactions in the catalytic triad and these hydrogen bonds.
8. The catalytic triad is not unique to chymotrypsin. The serine protease known as subtilisin also has the same catalytic triad and employs a similar mechanism.
9. Besides the catalytic triad, the enzyme has two other important sites to consider. The first is the oxyanion hole that stabilizes a tetrahedral intermediate that arises during the catalysis. The second, called as S1 pocket, is where the substrate binds. Both the oxyanion hole and the S1 pocket are adjacent to the active site (catalytic triad).
10. The S1 pocket determines a serine protease's specificity. The S1 pocket of chymotrypsin is hydrophobic and relatively large, allowing it to bind phenylalanine, for example. Remember that chymotrypsin cuts adjacent to phenylalanine (among other hydrophobic amino acids). The S1 pocket of trypsin, for example has a negatively charged group in the bottom, allowing it to bind to lysine or arginine.
11. Other proteases include cysteine proteases (use cysteine and histidine in the active site), aspartyl proteases (use aspartic acids and water in the active site) metalloproteases (use a metal ion - usually zinc - and water in the active site).
11. Carbonic anhydrase is an enzyme that catalyzes the joining of carbon dioxide and water to form carbonic acid.
12. A zinc ion (held in place by three histidines in the active site of carbonic anhydrase) plays an important role in the catalysis of the enzyme by binding a water molecule. A subsequent loss of a proton by water is necessary for catalysis. Notably, the enzyme has maximal activity at a high pH (where protons are easily removed) and a lower activity in an acidic pH (6.0).
13. The limiting step in the action of carbonic anhydrase is the abstraction of the proton from water. Buffers and/or bases help facilitate this and thus speed the reaction.
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