Exam 3 PDF

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1) Be able to explain the term irreversible inhibition. -

Dissociates very slowly from its target enzyme because it has become tightly to enzymes either covalently or noncovalently. Some irreversible inhibitors are important drugs, penicillin and aspirin. Ones that are covalently bonded are Powerful tools for elucidating the mechanisms of enzyme action. Modify functional groups, if treatment results in a loss of enzyme activity it suggests that the modified group is required for the activity Four categories: group specific, affinity labels, suicide inhibitors, and transition state analogs

2) Name the different categories of chemicals that cause irreversible inhibition and the mechanism for each with one example. -

Group-specific reagents: modify specific R groups of amino acids. Ex Diisopropylphosphofluoridate (DIPF). DIPF inhibits the proteolytic enzyme chymotrypsin by modifying only 1 of the 28 serine residues in the protein, implying that the serine residue is especially reactive. DIPF and similar compounds that bind and inactivate acetylcholinesterase are potent nerve gases.

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Substrate analogs/affinity labels: ( enzymatic steps lead to irreversible association) are molecules covalently modify active site residues and are structurally similar to an enzyme’s substrates. They are thus more specific for an enzyme’s active site than are group-specific reagents. EX. Tosyl-L-phenylalanine chloromethyl ketone (TPCK) is an affinity label for chymotrypsin. It binds at the active site and then reacts irreversibly with a histidine 57 residue

at the site, inhibiting the enzyme.

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Suicide inhibitors/mechanism based inhibition: chemically modified substrates. These molecules provide researchers with the most specific means of modifying an enzyme’s active site. Bind to the enzyme as a substrate. As catalysis occurs, the enzyme modifies the substrate, converting it into an irreversible inhibitor. - Ex. penicillin is a suicide inhibitor of an enzyme required for bacterial cell wall synthesis “Glycopeptide transpeptidase” - Penicillin is an antibiotic that consists of thiazolidine ring fused to a very reactive β -lactam ring Penicillin inhibits the formation of cell walls in certain bacteria such as S. aureus.

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Transition-state analogs: are potent inhibitors of enzymes. The formation of the transition state is crucial to enzyme catalysis.

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3) Given chemical structures of DIPF, TPCK, Penicillin and Pyrrole 2-carboxylic acid with or without their enzymes be able to identify the mechanism of irreversible inhibition or some related properties of the inhibitors. -

Group-Specific:

Affinity Labels:

Suicide Inhibitor:

4) In which of the above cases does the enzyme carry out a part of the reaction with the inhibitor as it would for a real substrate? - .Suicide inhibitors In which of the above cases is the inhibitor specific for the enzyme and in which case could it be non-specific? - Affinity is more specific. Group-specific is nonspecific. Be able to explain inhibition based on the structural similarities between the inhibitor and its natural substrate or the transition state of that substrate. -

5) Last year King Jong Nam was assassinated with a viscous liquid poison. What was that agent? Based on what we know about this category of poisons, what is its likely mechanism by which this poison must have killed him? -

AChE. group-specific. DIPF

6) Be able to explain how irreversible inhibitors of the enzyme chymotrypsin revealed important amino acid residues in its active site that carry out the hydrolysis of the peptide Bond. It is secreted in response to a meal. It cleaves peptide bonds selectively on the carboxyl-terminal side of the large hydrophobic amino acids such as tyrosine, phenylalanine, and methionine. The nucleophile becomes covalently attached to the substrate during catalysis, this is because chymotrypsin contains reactive serine residue ● Treatment with organoflurophosphates that modify serine residues, such as DIPF, was found to inactivate the enzyme irreversibility. Serine 195 was the only one affected and loss enzyme activity. The use of group-specific reagent DIPF=importance of one particular serine residue in catalysis

● Chymotrypsin is a proteolytic enzyme which breaks down proteins Be able to explain the term catalytic triad and the function of each member of the triad for chymotrypsin. - The catalytic triad is a group of three amino acids that are found in the active sites of some proteases involved in catalysis.

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*The catalytic triad ( left) converts serine 195 into a potent nucleophile ( right) Histidine 57: required for chymotrypsin activity. Acts as a base to catalyst to activate serine Serine 195: required for chymotrypsin activity. Provides nucleophile. The side chain is hydrogen bonded to the imidazole ring of histidine 57. Aspartate 102: stabilizes protonated histidine. The -NH group of the imidazole ring of the His-57 is hydrogen bonded to the carboxylate group of the Asp-102.

7) Be able to explain why the accumulation of colored product using a chromogenic substrate for chymotrypsin suggested a two step catalytic reaction instead a a simple one step reaction. - Studies with the chromogenic substrate reveal that catalysis by chymotrypsin occurs in two stages: a rapid step (pre-steady state) and a slower step (steady state) - The steps in catalysis are explained by the rapid formation of an acyl-enzyme intermediate and a slower release of the acyl component to regenerate free enzyme - Two steps steps are explained by the formation of a covalently bound enzymesubstrate intermediate. First, the acyl group of the substrate becomes covalently attached to serine 195 of the enzymes as p-nitrophenolate is released. The enzymeacyl-group complex is called the acyl-enzyme intermediate. Second, the acyl-enzyme intermediate is hydrolyzed to release the carboxylic acid component of the substrate and regenerate the free enzyme.

8) Be able to recognize the key steps in the catalysis and given a particular step/s of the reaction mechanism be able to identify the correct description from a set of alternatives using chymotrypsin as an example. What is an oxyanion hole in chymotrypsin and what role does it play in the catalysis? A region of the active site, stabilizes the tetrahedral reaction intermediate. Interactions with the NH groups in the oxyanion hole help to stabilize the tetrahedral intermediate. These interactions contribute to the binding energy that helps stabilize the transition state that proceeds the formation of the tetrahedral intermediate. Which intermediates are unstable? Tetrahedral-intermediate form bears a negative charge on the oxygen atom derived from the carbonyl group.

9) Be able to identify whether histidine acts as a base or as an acid in given steps. It depends on the step you are on ( Base, acid, base, acid)

10) Be able to explain why chymotrypsin cleaves after large hydrophobic or aromatic residues based on the understanding of the amino acid residues in the S1 pocket. -

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A pocket enriched for hydrophobic residues is best occupied by a substrate that has a large aromatic or large hydrophobic residue followed by a peptide bond that is close to catalytic serine 195. The binding of an appropriate side chain into this pocket positions the adjacent peptide bond into the active site for cleavage. The specificity of chymotrypsin depends almost entirely on which amino acid is directly on the amino terminal side of the peptide bond to be cleaved.

Chapter 14 1. Be able to explain the importance of food.

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Eating is a basic need of all organisms that relieves hunger and provides energy. Turning a meal into cellular biochemical. We eat for growth and maintenance.

2. What are the constituents in our food that must be broken down into smaller components before being absorbed by our body? -

Proteins: digested to amino acids by proteolytic enzymes (proteases) secreted by the stomach and pancreas. Lipids: are converted into fatty acids by lipases secreted by the pancreas. Polysaccharides: such as starch, are cleaved into monosaccharides by alpha-amylase from the pancreas and to a lesser extent in saliva.

3. Which common chemical reaction mechanism is employed by the enzymes that break down food into smaller chemical units? -

A diverse set of hydrolytic enzymes. Hydrolases; cleave their substrates by the addition of a molecule of water.

4. Name an enzyme present in saliva? Which molecule does it help break down? Is this breakdown of the macromolecule to a significant extent or to a very small extent and why? -

Amylase, breaks down starch. Lesser extent. Because of the short duration of time that food is in our mouth.

5. Why must we chew and grind food to make an aqueous slurry? -

Chewing converts the meal into a slurry that is more readily attacked by hydrolytic enzymes.

6. Where does the digestion of proteins begin? -

In the stomach

What is the pH of this part of the digestive system? -

Highly acidic pH=1.5 to 3.5

What is the effect of this pH on the proteins from food? How does this help the enzyme pepsin to carry out the hydrolysis of proteins? -

The highly acidic environment of the stomach denatures proteins, making them more susceptible to digestion by proteolytic enzymes, such as the stomach enzyme pepsin.

7. What is the nature of the active transport protein that causes the contents of the stomach to be highly acidic? -

K+/H+ ATPase

8. In disease such as GERD, how can we block the function of the above active transporter irreversibly using specific inhibitor?

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GERD can be treated with inhibitors of the K+/H+ ATPase such as omeprazole, a common treatment for GERD is to irreversibly inhibit the proton pump. One such inhibitor Omeprazole is converted into sulfenic acid by the stomach acid, which rearranges to yield sulfonamide. Sulfonamide irreversibility modifies a cysteine residue on the pump.

9. In what form is pepsinogen produced by the stomach cells? Does pepsinogen have any catalytic activity at all? How is pepsin produced from pepsinogen? Which is more effective at carrying out the hydrolysis of proteins pepsinogen or pepsin? -

Pepsin (more effective because it’s the active form). Pepsinogen is an inactive form of pepsin. It has a small amount of enzyme activity and can activate itself in some degree in an acidic environment. Pepsin is more effective at hydrolyzing proteins.

10. Where does the food pass into from the stomach? How are the food contents from the stomach neutralized as it enters the intestine? Which hormone helps in this process? -

The movement of food from the stomach to the intestine stimulates the secretion of two key hormones of the small intestine. - Secretin causes the release of sodium bicarbonate, which neutralizes the stomach acid. The low pH of the food stimulates the cells of the small intestine. - Cholecystokinin (CCK) stimulates the release of digestive enzymes from the pancreas as well as the secretion of bile salts from the gallbladder.

11. Name another hormone released by the intestinal cells in response to food? Which two target organs respond to this hormone? What does each organ secrete in response to this hormone? Be able to recognize the key components shown in figure depicting this hormonal control.

12. Most enzymes secreted by the pancreas are inactive and known as pro- enzymes or Zymogens? How are they converted into their active forms in the intestine? -

Activated by proteolytic cleavage. When a part of the inactive precursor is proteolytically cleaved. The enzyme enteropeptidase (enterokinase), secreted by the epithelial cells of the small intestine, activates the pancreatic zymogen trypsinogen to form trypsin which in turn activates the remaining pancreatic zymogens.

Which enzyme cleaves and activates trypsinogen to trypsin? -

Enteropeptidase, secreted by intestinal cells, converts inactive trypsinogen into active trypsin. trypsin , in turn, activates the other proenzymes.

What are the different pancreatic pro-enzymes that tryspin cleaves and activates?

Be able to explain why trypsin deficiency may cause more severe disease as compared to deficiency of some other pancreatic enzymes that digest proteins. -

Because since trypsin activates many other zymogens it will have a bigger effect on the body if there is a lack of trypsin

13. What are the various products of protein digestion and how are they transported into the intestinal cells? How are these components further delivered to the blood? -

Proteins are digested into amino acids and small oligopeptides. The amino acids are absorbed by transporters. Peptidases on the surface of intestinal cells cleave the oligopeptides into di and tripeptides which are transported into the intestinal cells and degraded into amino acids. The amino acids are subsequently released into the blood by antiporters.

14. What are the amino acids enriched in proteins generally referred to as gluten? -

Glutamine and proline

What disease is caused if an individual is susceptible to the peptides derived from gluten? What is a consequence of chronic inflammation due to gluten derived peptides? What must such individuals ensure so that they do not suffer? -

Celiac disease, or gluten enteropathy, is an intestinal inflammatory disorder that results because susceptible individuals are unable to digest certain proteins from wheat, rye, and barley. The gluten-derived peptides generate an inflammatory response that damages the intestinal lining and impairs nutrient absorption.

15. Which form of carbohydrate is most abundant in our diet? -

Starch

Name two disaccharides that we consume often in coffee (café au lait)? -

Sucrose and lactose

16. How do we breakdown complex carbohydrates (eg starch, glycogen)? How large are these polysaccharides? Which bonds exist between the monosaccharide units in such carbohydrates? -

alpha -amylase cleaves alpha-1,4 bonds of starch but not alpha-1,6 bonds

17. Which bond is hydrolyzed by alpha-amylase? What are the two sources of alphaamylase in our digestive system? -

The 1-4 bonds of starch are hydrolyzed by the alpha amylase. Alpha amylases can be found in saliva and secreted from the pancreas.

18. If starch or glycogen is broken down by amylases, what are the products formed? How do we carry out the further digestion of these products?

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Di and trisaccharides maltose and maltotriose, and limit dextrin. These products are furthered digested via enzymes: ● Maltase converts maltose into glucose. ● Alpha-glucosidase digests maltotriose and other oligosaccharides. ● Alpha dextrinase digests limit dextrin into simple sugars.

19. How do we breakdown the two disaccharides that are present in coffee with milk? -

Through enzymes, the two disaccharides that are present are sucrose and lactose. Each can be degraded into simpler forms by sucrase and lactase enzymes respectively. These two enzymes are found on the surfaces of intestinal cells.

20. How do uptake the various monosaccharides produced by the action of other enzymes or may be directly a part of our diet into our body? Discuss the role of various transport proteins in the intestinal cells. -

Glucose and galactose are transported into intestinal epithelial cells through secondary active-transport process carried out by the sodium-glucose linked transporter (SGLT). Fructose is diffused across the cell membrane by a transporter called GLUT5. GLUT2 releases these three monosaccharides into the bloodstream.

21. Why is digestion of lipids an issue as compared to digestion of carbohydrates or proteins and how does our body solve this problem? -

It is an issue because lipids are not soluble in water, the medium where digestive enzymes are present. Lipids are prepared for digestion in the stomach. The stomach converts the lipids into an emulsion (a mixture of lipid droplets and water) by grinding and mixing. Once the lipids leave the stomach, the lipids undergo further emulsification by bile salts. Bile salts are amphipathic molecules that are inserted into the lipid droplets. This creates triacylglycerols that can be easily degraded into fatty acids and monoacylglycerol by enzymes called lipases. These products are absorbed by the epithelial cells.

22. Where does the preparation for lipid digestion first begin? -

Stomach. Emulsion is a mixture of lipid droplets and water.

23. Why are secretions from the gallbladder important to help with digestion of lipids? -

Help making the triacylglycerols readily digested. Bile salts, secreted by the gallbladder, insert the lipid droplets, rendering them more accessible to digestion by lipases.

24. Which hormone from the small intestine is important for stimulating these secretions? -

Cholecystokinin

25. What is the fate of triacylglycerols in the intestine? Which enzyme leads to its hydrolysis and what are the products of this hydrolysis? How are the digestion products eg fatty acids carried to the intestinal cell? How are the fatty acids and monoacyl

glycerol organized in micelles? (which parts face aqueous phase and which face the interior of the micelle) -

Ionized fatty acids form micelles. The digestion products are carried as micelles to the intestinal epithelium cells for absorption.

26. Liver produces the bile secretions that ultimately reach the gallbladder from where they are released into the intestine. What causes steatorrhoea, a condition that leads to the appearance of large amounts of fats such as stearic acid in the feces? -

If production of bile salts is inadequate due to liver disease, large amounts of fats are excreted in the feces. This is called steatorrhea, after stearic acid, a common fatty acid.

27. How are fatty acids and monoacylglycerols transported into the intestinal cell? How and where are triacylglycerols resynthesized? What is the further fate of the TAG that are resynthesized in the intestinal cell and in what form would it leave the intestinal cell? What are the other components associated with the TAGs as they exit the intestinal cell to enter the lymph before they finally reach the blood. -

Into the intestinal cells by membrane proteins such as fatty-acid-binding protein (FABP). Once inside the cell, fatty-acid-transport proteins (FATP) ferry them to the cytoplasmic face of the smooth endoplasmic reticulum (SER), where the triacyglycerols are resynthesized from fatty acids and monoacyglycerol. After transport into the lumen of the SER, the triacyglycerols associate with specific proteins and a small amount of phospholipid and cholesterol to form lipoprotein transport particles called chylomicrons.

How does the blood appear after a lipid rich meal? -

The blood appears milky because of the high content of chylomicrons.

28. How do the lipids in the chylomicrons absorbed into other cells of the body (eg muscle ) from blood? -

The chylomicrons eventually enter the blood so that the triacyglycerols eventually enter the blood so that the triacyglycerols can be absorbed by tissues. These particles bind to the membrane-bound lipoprotein lipases, primarily at adipose tissue and muscle, where the triacyglycerols are once again degraded into free fatty...