MCM 2-37 Enzyme Regulation and Clinical Application Flashcards Preview

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Flashcards in MCM 2-37 Enzyme Regulation and Clinical Application Deck (38)
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1

allosteric enzymes frequently operate at..

control points in metabolic pathways
ex)feedback inhibition

2

allosteric enzymes do not follow michaelis mentron kinetics (TF)

K0.5?

True

allosteric enzymes show and S-shaped curve rather than rectangular hyperbola

K0.5 = [S] giving half maximal activity

3

allosteric modulators can affect the

either K.05 or Vmax and be activators or inhibitors

4

allosteric regulation

substances (allosteric modulators) binding the enzyme somewhere other than the active site and modifying its activity

5

heterotrophic vs homotrophic effector

heterotrophic effector - when the allosteric modulator is NOT the substrate for the enzyme

homotrophic - when the allosteric modulator IS the substrate for the enzyme

6

ATCase example

allosteric enzyme example

catalyzes initial step in pyrimidine (CTP) synthesis pathway

CTP is an allosteric inhibitor, working through negative feedback mechanism shifting the enzyme equilibrium towards less active T state. (shift curve to left) by preferentially binding and stabilizing the low-affinity conformation of ATCase (T state)

ATP is an allosteric activator, shifting the enzymes equilibrium towards the more active R state by binding and stabalizing the high affinity conformation of ATCase

7

reversible covalent modification


example?

mechanism involves substances being covalently bound to the enzyme in a reversible manner (phosphorylation, methylation, ubiquitination) which can cause in the enzyme
conformational change altering catalysis
altering cellular localization of the enzyme
altering interactions with other enzymes

glycogen phosphorylase is an example of this type of enzyme. Phosphorylation activates it, dephosphorylation deactivates it.

8

irreversible covalent modification

involves substances being permanently covalently bound to the enzyme. enzymes regulated this way are synthesized in inactive form and then irreversibly activated where/when needed.

ex) zymogens

9

Zymogens

examples?

example of irreversible covalent modification

inactive precursors to proteases that are cleaved and activated by other proteases
-digestive enzymes in stomach/pancreas (pepsin, chymotrypsin)
-clotting cascade enzymes (thrombin)
-caspases (involved in programmed cell death; caspase cascade)

10

protein-protein interactions

examples?

involves specific proteins interacting with enzymes to alter their activity.

protease inhibitors, for example, interact with proteases and inactivate them. otherwise would be permanently activated.

anti-thrombin III inactivates thrombin and a number of proteases in the clotting cascade and arrests clotting

alpha-1-antitrypsin (AAT) inhibits elastase in the lungs.

11

signaling cascade enzymes (like PKA and calmodulin) are generally regulated via

protein-protein interactions

12

digestive enzymes are synthesized as

zymogens (protease precursors) stored in pancreas. response to food availbaility signals, released into duodenum where enteropeptidase cleaves trypsinogen to trypsin which cleaves others (proteolytic cascade)

13

anticoagulants

mimic vitamin K, are competitive inhibitors to the vitamin-K-dependant enzyme, they mimic vitamin K deficiency. preventing/slowing clotting

14

What is TPA and when given?

TPA - serine protease that hydrolyzes plasinogen to plasmin which break up fibrin clots. combat one protease cascade with another.

administered therapeutically for heart attack and stroke

15

anti-thrombin (AT III) deficiency

can cause?

treatment?

autosomal dominant interited genetic disease, patients have one missing or defective copy of gene

excessive clotting (Thrombosis)
blood clots form inappropriately (in legs or lungs) after serious injury or oral contraceptive use. can be fatal


treated with long term anti-coagulants

16

alpha1-antitrypsin deficiency

caused by?

loses ability to do what?

too much active elastase in lung eventually causes emphysema due to bronchiol digestion (COPD)

diagonosed under ocontions that recruit elastase producting macrophages to lungs (smoking, lung infection)

patient can't inhibit elastase and experiences shortness of breath

severe deficiency is often occompanied by liver disease (misfolding in ER)

17

protease inhibitors vs protein-protein interactions

protease inhibitors like serpine often inhibit irreversibly

enzyme activity can be regulated reversibly by interactions with other proteins

18

signal transduction cascades are often regulated by


examples?

protein-protein interactions

protein Kinase A - regulated by interaction of cAMP with regulatory subunits
-cAMP releases regulatory subunits and activates catalysis

calmodulin - shows Ca+2 dependant interactions with multiple enzymes

19

need to regulate enzymes on different _____

substrate availability determines _____

time scales

enzymatic activity - big changes below Km, not much if new Vmax

20

product inhibition

allosteric control

covalent modification

product inhibition - reaction product goes back and inhibits, change in vmax or Km, immediate

allosteric - pathway end product can change Vmax or K0.5, immediate

Covalent modification - requires another enzyme, fast but not immediate, change in vmax or Km

21

Enzymes as diagnostic tools can do what?

diagnostic measurement of enzyme levels

measurement of substrate or metabolite levels

diagnosis of tissue damage or tumors by isozyme distribution

22

after a blood vessel injury, the following three rapid responses are needed for maintainece of blood volume


what do they all have in common?

1. rapid activation of blood coagulation - zymogen cascade activating series of serine proteases. each protease can activate many other target proteases, amplifying the signal

2. localization of clot to site of injury - vitamin -K dependant pathway modulates glutamate residues on several of the cascades serine proteases allowing Ca+2 to bridge the modified proteases and then membrane at the site of injury. This is a reversible covalent modification.

3. rapid termination after clot formation to prevent thrombosis - opposing cascade involving different serine proteases resulting in clot hydrolyzation

these are all regulated by irreversible covalent modifications

23

enzyme assays


performed under what conditions?

measures enzyme activity at a saturated [S] where Vmax is achieved so that the rate of reaction is linear to the amount of enzyme present.

lactate dehydrogenase assay - detecting sample absorbance at 340nm, NADH (product) is more absorbent then NAD) reactant, at this wavelength.


excess substrate (lactate and NAD)

24

coupled enzyme assay

when direct measurement of product appearance or substrate disappearance is not possible

second enzyme utilized that uses product of first enzyme as its substrate.

product of second enzyme is detected and used to indirectly determine the intiial enzyme activity.

ALT;SGPT enzyme assay is an example
-second enzyme produced NADH when it reacts with alanine (product of SGPT) and NAD. NADH is detected spectrophotometrically.

add everything in excess except enzyme #1 and products of both enzymes

25

enzymatic metabolite assay


benefits?

disadvantage?

example?

detect small molecules in complex mixture. large amount of enzyme added so that all substance can be converted into product in short amount of time.

amount of product formed is detected directly or indirectly

specific, fast, gentle on sensitive substrates

other factors in mixture can interfere with enzyme activity


blood glucose level measurements
1. couple enzyme assay converts glucose to NADPH, which is easily detectable and proportional to the glucose substrate.
2. colorless dye added, reduced to colored form in prescence of NADPH.
3. allows for coloimetric measurement of glucose concentration

26

enzymatic tissue damage assays

enzymes detected in serum that serve no physiological purpose there are a hallmark of tissue damage or tumor growth.

-detected by measuring level of activity, timing appearnace of activity, or precence of tissue specific Isozymes

27

isozymes

different forms of an enzyme that carry out same reaction. some are tissue specific

28

ALT (alanine aminotransferase, ALT or SGPT) enzyme in plasma

viral hepatitis

29

lactate dehydrogenase isozyme 5 in plasma?

liver diseases

30

amylase in plasma

acute pancreatitis

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