sakai-Enzymes Flashcards

Question

Discuss the enzymatic activities for pepsin, trypsin and alkaline phosphatase at different pH levels! What can be the reason (related to pH) for low activity of pepsin or for low activity of trypsin in some individuals?

Answer 1

In these experiments, the velocity is measured under constant enzyme and substrate conditions, only the pH is different in the vials . Pepsin, is an enzyme that is active in the stomach lumen and has an acidic pH optimum (pH 2) Trypsin, an enzyme that is active in the duodenum, needs a more neutral pH for activity. Alkaline phosphatase was named after the fact that it cleaves phosphate groups and has an alkaline pH optimum. An increase of the pH of the stomach lumen by antacids can reduce the activity of pepsin. It can also reduce the activation of pepsinogen to pepsin. Also, the generation of gastric acid can decline in the elderly leading to less pepsin activity. The acid chyme (from the stomach) needs to be neutralized in the duodenum to generate a pH that is favorable for trypsin activity (pH 6-7). A deficiency of the release of bicarbonate from the pancreas can lead to less trypsin activity in the duodenum due to an acidic pH. [as examples, less bicarbonate reaches the duodenum in patients with deficiency of secretin or in patients with cystic fibrosis]

Answer 2

Changes of concentrations of substrates or of products Modulation of enzyme concentration Covalent modification of enzymes Modulation of a metabolic pathway

Answer 3

The velocity of an enzyme increases with substrate concentration as more product can be formed. If the substrate is not available, then the product cannot be formed. [Sometimes the substrate for a pathway has to be generated in the correct cell compartment. As an example, the cytosolic enzyme for the regulated step of fatty acid synthesis needs acetyl CoA in the cytosol. When this substrate is not present, then the pathway cannot be performed]

Answer 4

Covalent enzyme modification is faster than enzyme induction. Covalent enzyme regulation acts on the present enzyme, whereas enzyme induction acts via an increased amount of enzyme and needs the processes of transcription and translation in order to synthesize a new enzyme.

Answer 5

The catalysis of a protein kinase is irreversible. The enzyme is a transferase that transfers a phosphate group from ATP to the protein. Yes, the catalysis of a protein phosphatase is irreversible. The enzyme cleaves a phosphate group from the protein. This is important as it allows hormonal control that can assure that the protein stays phorphorylated or on the other hand, following the action of another hormone, the enzyme is reversed to its dephosphorylated form.

Answer 6

The enzyme regulation via phosphorylation/dephosphorylation in itself is reversible. Two different enzymes are needed, a protein kinase and a protein phosphatase. As an example: When the enzyme is phosphorylated by a protein kinase, then a conformational change takes place which can either activate or inhibit the enzyme. When the phosphorylated enzyme is changed back to the unphosphorylated form of the enzyme by a protein phosphatase, then the original enzyme conformation is again obtained.

Answer 7

Yes. Increase in temperature leads to an increase in velocity until the denaturation of human enzymes starts at high fever. On the other hand, heart surgery is often performed with cardiac arrest solutions and cooling of the heart in the open chest with ice in order to reduce enzyme activity and save energy.

Answer 8

Enzyme induction means more enzyme synthesis than normal, often following as response to a hormone. This regulation uses the modulation of the concentration of the available enzyme. Glucagon signals low blood glucose and it leads in the liver to induction of key enzymes for gluconeogenesis. This pathway forms and releases glucose into the blood and more enzymes can lead to more products. Induction of enzymes of glycolysis is not wanted after glucagon action, as glycolysis in the liver reduces blood glucose. Insulin signals high blood glucose levels and leads in the liver to induction of key enzymes for glycolysis.

Answer 9

Proteolytic activation leads to a covalent modification of the proenzyme by cleavage of a protein part and to refolding of the now smaller enzyme. This activation is irreversible and the activated enzymes can only be inactivated by degradation of the active enzyme or sometimes by specific inhibitor proteins. The advantage of this regulation is that an irreversible and highly specific cascade mechanism can be used involving specific enzymes.

Answer 10

Blood clotting factors Digestive proteases and pancreatic phospholipase 1 Blood clotting has to be performed fast in case of bleeding. The inactive blood clotting factors are already available in the blood, but their activation shall be highly specific and controlled. In the blood clotting cascade, there is a logical activation of finally prothrombin to thrombin for the formation of the fibrin clot. The enzyme thrombin has a small window for its action before it gets inactivated by antithrombin III. [Blood clotting has to be performed fast and locally directed and on the other hand it has also to be stopped. Heparin accelerates the inactivation of thrombin and is often used as drug during surgery to prevent blood clotting.] 2 The digestive pancreatic proteases and phospholipase are powerful enzymes and meant to degrade dietary proteins and phospholipids. They are synthesized inside of pancreatic cells in their inactive forms and are meant to be later activated only in the lumen of the duodenum. As example, the inactive trypsinogen reaches via the pancreatic juice the duodenum and is there proteolytically activated. The enzyme enteropeptidase is highly specific to cleave trypsinogen to trypsin. This local separation normally prevents premature formation of trypsin in the pancreatic cells. This is important as trypsin itself is able to perform proteolytic activation of digestive proenzymes. [After the dietary protein is digested, the digestive enzymes, which are proteins, will be degraded by their fellow digestive proteases and the amino acids are taken up into intestinal mucosal cells in addition to the amino acids from dietary proteins.]

Answer 11

A homotropic effector is “one of the same” of the molecules encountered by the allosteric enzyme. The substrate for the enzyme can act as homotropic effector. A heterotropic effector is a different kind of molecule, it is not the substrate and it is not the product of the allosteric enzyme. A heterotropic effector is formed by another enzyme. The allosteric enzyme has a very specific binding site for the respective heterotropic effector, which could lead to feedback inhibition or feed-forward activation of pathways, dependent on the molecule. [the regulated allosteric enzyme “wants to be informed about the metabolic situation of the total pathway”]

Answer 12

Yes, an allosteric enzyme can have binding sites for a homotropic effector, like the substrate, and in this case, the binding site is in the active site. It can also have in addition different binding sites for several heterotropic effectors

Answer 13

Allosteric enzymes can perform a conformational shift after binding of heterotropic effectors. For example, a pathway can be down-regulated when the end product (or a related compound) of the respective pathway binds to a specific binding site of the allosteric enzyme at the rate-limiting step. This can induce a conformational shift which leads to less activity of the enzyme, (often the affinity for the substrate is reduced by the conformational change that follows after the binding of the respective heterotropic effector.)

Answer 14

K 0.5 gives similar information as Km does: a small K 0.5 indicates a high affinity of the allosteric enzyme for the respective substrate. Allosteric enzymes can make a conformational shift, like in case of feedback inhibition, after binding of a specific heterotropic effector. In this new conformation with the feedback regulator bound, the affinity of the allosteric enzyme for the substrate can decrease, leading to a larger K 0.5

Answer 15

When an allosteric enzyme is feedback inhibited, and when as the result K 0.5 is changed, then the substrate concentration at half Vmax , [which is K 0.5 ] is larger than in the normal reaction. The enzyme has less affinity for the substrate due to a conformational shift and the pathway is feedback inhibited. [the curve is shifted to the right} Please DO NOT mix up feedback regulation of specific allosteric enzymes with competitive inhibition of enzymes. !!! [both lead to a higher substrate concentration to reach half Vmax, but both are totally different inhibitions] A competitive inhibitor is mostly a synthesized drug and competes with substrate binding. The affinity of the enzyme for the substrate is not changed, (Km is a constant) but we find a larger apparent Km, which is the value of a larger substrate concentration needed to reach half Vmax due to the competition for formation of the ES complex. On the contrary, a feedback regulation is achieved by a natural compound which has already a specific binding site at the regulated allosteric enzyme and this molecule does not compete with the substrate binding. In this case of feedback inhibition, the actual affinity of the enzyme for the substrate is reduced due to a conformational shift of the enzyme and K0.5 is increased

Answer 16

Feed-forward activation of an allosteric enzyme leads to a conformational shift after binding of the respective heterotropic effector In this case, the affinity of the enzyme for the substrate is increased, and half Vmax is reached at lower substrate concentration than for the not feed- forward activated enzyme. [ note: feed-forward activation of an allosteric enzyme is different than the cooperative substrate binding where the substrate acts as a homotropic effector leading to a sigmoidal curve. Feed-forward activation shifts the already sigmoidal curve to the left.]

Answer 17

Purified enzymes from bacteria living in hot springs can be used for research as they tolerate high temperatures during experiments. The drugs acetaminophen and ibuprofen inhibit reversibly COX and are used for pain treatment and reduction of inflammation. Aspirin as irreversible inhibitor can be used in long term low dose to reduce blood clotting.