Test 2: Diagnostic Enzymology Flashcards
Enzymes are mainly _________ that facilitate biochemical reactions.
proteins
-ribozymes: RNA splicing
Purpose of measuring enzymes?
-Can measure the ACTIVITY of these enzymes in the blood to ascertain whether these organs have been or are being damaged.
-Abnormal serum enzyme levels are found in various diseases and
inflammation
What are the types of inhibition for enzyme activity?
1) Competitive inhibition
2) Allosteric inhibition
3. pH
4.Temperature
5.Presence of Inhibitor
- Inactive or less active precursor of enzyme
-Proteolytic modification required to be activated
Proenzyme (Zymogen)
Examples of Proenzyme (Zymogen)?
Angiotensinogen,
trypsinogen, pepsinogen,
chymotrysionogen, prolipase
Enzymes work by converting a substrate into a product via an ______________.
enzyme-substrate complex
Km =
(K-1 + K2) / K1
= Dissociation / Association
How dose enzymes speed up reactions?
-by lowering activation energy
-Increasing “rate constant”
-increasing “Substrate specificity” (or Substrate concentration)
Endergonic or exergonic:
If delta G is positive? If negative?
Endergonic
Exergonic
Reaction rate = K[S]x[S]y
What is the rate constant?
K
What are the factors influencing the rate of reaction?
1.Enzyme concentration
2.Substrate Concentration
increased [substrate], the rate of reaction ___________.
increases
- But [substrate] too high, enzyme Saturation
The shape of the protein affects its _________
activity
-Anything that alters the conformation of the
protein/enzyme will have an impact on its activity.
enzymes require a pH of 7-8. What are some exceptions?
alkaline phosphatase, acid phosphatase, pepsin
How are single (end) point assays done?
- Incubate sample with substrate for a period of time
- Measure the end absorbance (O.D.)
- Calculate enzyme level by comparing to the [STD]
How are kinetic assays done?
- Incubate sample with substrate
- Measure the absorbance over time at certain increments
- An average change in absorbance (product formation) is
used to calculate “enzyme activity”
IU or U =
The amount of enzyme that produces 1μM of product per minute under standard conditions
Use _________ to convert absorbance to μM
molar absorptivity
How do you calculate enzyme activity?
Δ absorbance / molar absorptivity x dilution factor
total volume / sample volume