MCM 2-37 Enzyme Regulation and Clinical Application

Question 1

allosteric enzymes frequently operate at..

Answer 1

control points in metabolic pathways

ex)feedback inhibition

Question 2

allosteric enzymes do not follow michaelis mentron kinetics (TF)

K0.5?

Answer 2

True

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

K0.5 = [S] giving half maximal activity

Question 3

allosteric modulators can affect the

Answer 3

either K.05 or Vmax and be activators or inhibitors

Question 4

allosteric regulation

Answer 4

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

Question 5

heterotrophic vs homotrophic effector

Answer 5

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

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

Question 6

ATCase example

Answer 6

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

Question 7

reversible covalent modification

example?

Answer 7

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.

Question 8

irreversible covalent modification

Answer 8

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

Question 9

Zymogens

examples?

Answer 9

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)

Question 10

protein-protein interactions

examples?

Answer 10

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.

Question 11

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

Answer 11

protein-protein interactions

Question 12

digestive enzymes are synthesized as

Answer 12

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)

Question 13

anticoagulants

Answer 13

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

Question 14

What is TPA and when given?

Answer 14

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

Question 15

anti-thrombin (AT III) deficiency

can cause?

treatment?

Answer 15

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

Question 16

alpha1-antitrypsin deficiency

caused by?

loses ability to do what?

Answer 16

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)

Question 17

protease inhibitors vs protein-protein interactions

Answer 17

protease inhibitors like serpine often inhibit irreversibly

enzyme activity can be regulated reversibly by interactions with other proteins

Question 18

signal transduction cascades are often regulated by

examples?

Answer 18

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

Question 19

need to regulate enzymes on different _____

substrate availability determines _____

Answer 19

time scales

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

Question 20

product inhibition

allosteric control

covalent modification

Answer 20

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

Question 21

Enzymes as diagnostic tools can do what?

Answer 21

diagnostic measurement of enzyme levels

measurement of substrate or metabolite levels

diagnosis of tissue damage or tumors by isozyme distribution

Question 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?

Answer 22

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

Question 23

enzyme assays

performed under what conditions?

Answer 23

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)

Question 24

coupled enzyme assay

Answer 24

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

Question 25

enzymatic metabolite assay

benefits?

disadvantage?

example?

Answer 25

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

Question 26

enzymatic tissue damage assays

Answer 26

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

Question 27

isozymes

Answer 27

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

Question 28

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

Answer 28

viral hepatitis

Question 29

lactate dehydrogenase isozyme 5 in plasma?

Answer 29

liver diseases

Question 30

amylase in plasma

Answer 30

acute pancreatitis

Question 31

lipase in plasma

Answer 31

acute pancreatitis

Question 32

creatine kinase in plasma

Answer 32

muscle disorders and myocardial infarction

Question 33

acid phosphatase in plasma

Answer 33

metastatic carcinoma of prostate

Question 34

alkaline phosphatase isozymes in plasma

Answer 34

various bone disorders and obstructive liver disease

Question 35

pancreatic trypsin inhibitor

Answer 35

inhibits inappropriately activated digestive proteases in pancreas

Question 36

ATIII (anti-thrombin)

Answer 36

inactivates thrombin and other proteases of clotting cascade, arrests clotting

Question 37

alpha-1-antitrypsin (AAT)

Answer 37

inhibits elastase in the lung

Question 38

TPA

Answer 38

“clot buster”. “plasminogen activtor”. helps activate plasminogen to plasmin, which then binds to a fibrin clot and breaks it up

administered close to heart attack or stroke