MCM 2-37 Enzyme Regulation and Clinical Application Flashcards
allosteric enzymes frequently operate at..
control points in metabolic pathways
ex)feedback inhibition
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
allosteric modulators can affect the
either K.05 or Vmax and be activators or inhibitors
allosteric regulation
substances (allosteric modulators) binding the enzyme somewhere other than the active site and modifying its activity
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
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
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.
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
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)
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.
signaling cascade enzymes (like PKA and calmodulin) are generally regulated via
protein-protein interactions
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)
anticoagulants
mimic vitamin K, are competitive inhibitors to the vitamin-K-dependant enzyme, they mimic vitamin K deficiency. preventing/slowing clotting
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
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
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)
protease inhibitors vs protein-protein interactions
protease inhibitors like serpine often inhibit irreversibly
enzyme activity can be regulated reversibly by interactions with other proteins
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
need to regulate enzymes on different _____
substrate availability determines _____
time scales
enzymatic activity - big changes below Km, not much if new Vmax
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
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
after a blood vessel injury, the following three rapid responses are needed for maintainece of blood volume
what do they all have in common?
- rapid activation of blood coagulation - zymogen cascade activating series of serine proteases. each protease can activate many other target proteases, amplifying the signal
- 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.
- 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
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)
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
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
- couple enzyme assay converts glucose to NADPH, which is easily detectable and proportional to the glucose substrate.
- colorless dye added, reduced to colored form in prescence of NADPH.
- allows for coloimetric measurement of glucose concentration
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
isozymes
different forms of an enzyme that carry out same reaction. some are tissue specific
ALT (alanine aminotransferase, ALT or SGPT) enzyme in plasma
viral hepatitis
lactate dehydrogenase isozyme 5 in plasma?
liver diseases
amylase in plasma
acute pancreatitis
lipase in plasma
acute pancreatitis
creatine kinase in plasma
muscle disorders and myocardial infarction
acid phosphatase in plasma
metastatic carcinoma of prostate
alkaline phosphatase isozymes in plasma
various bone disorders and obstructive liver disease
pancreatic trypsin inhibitor
inhibits inappropriately activated digestive proteases in pancreas
ATIII (anti-thrombin)
inactivates thrombin and other proteases of clotting cascade, arrests clotting
alpha-1-antitrypsin (AAT)
inhibits elastase in the lung
TPA
“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
