Lecture 1: Enzymology Flashcards
What are enzymes?
Proteins with catalytic properties
What are the 6 different classes of enzymes?
Oxidoreductase
Transferase
Hydrolase
Lyase
Isomerase
Ligase
What is enzymology used for in the clinical scene?
- Application of enzyme analysis in diagnosis or treatment of disease
- Determine enzyme deficiency
- Indicate tissue damage
- Indicate organ failure
What are some enzymes used to determine liver function, pancreas function, and general cell damage?
Liver function: AST, ALT, ALP, GGT
Pancreas: Amylase, Lipase
General Cell Damage: LDH
What is the protein structure of an enzyme and what does it need to function properly?
3D structure required to be functional
Multiple 3D structures may be required for proper enzyme activity
What are isoenzymes?
Enzymes that are related but differ based on aa sequence
What properties change due to the different aa sequences in isoenzymes?
- Physical and biochemical properties
- Immunologic properties
- Physiological role
What are the clinical relevant isoenzymes?
LDH, CK, Amylase and ALP
What type of lab tests are used to assess isoenzymes?
- Electrophoresis - size and charge differences
- Chromatography - size, charge and polarity
- Immunoassay - aa sequence
What are macroenzymes? What are some examples?
High MW complexes formed when circulating enzymes cross linked to Ig (Type 1)
OR formed through spontaneous polymerization
Examples: Macro-amylase, macro-CK, macro-ALP, macro-LDH
What are some cons of testing for macroenzymes?
- Longer clearance, and significant elevation in plasma
- Clinically benign - not indicative of injury/disease
How do we express enzyme activity?
U/L = unit of activity/volume
where U is the quantity of enzyme that catalyzes 1 umol substrate/min
What is the purpose of the Michaelis-Menten Curve?
- Assess velocity at varying substrate concentrations
- Determine Km
- Identify possible inhibition at high concentration
What is the Lineweaver-Burk Transformation?
Inverse transformation of velocity & substrate concentration
Differentiate between a 0 order and 1st order reaction.
Zero order
1. Independent to substrate concentration
2. Substrate excess & all active sites of enzymes are complexed
3. Rate of reaction is based on enzyme activity and/or concentration
First order
1. Proportional & dependent to substrate concentration
2. Enzyme concentration is constant
3. Rate of reaction is based on substrate concentration
How does the creatine kinase assay work? Which reactions are 0 order and 1st order? How do you know?
Creatine phosphate + ADP ➔ (CK) ➔ Creatine + ADP
0 order rxn bc has substrate excess
Want to establish relationship between CK and creatine
Glucose + ATP ➔ (HK) ➔ G6P + ADP
1st order rxn bc enzyme excess
G6P + NADP ➔ (G6P-DH) ➔ gluconate-6-P + NADPH + H
1st order rxn
Rate of formation of NADPH is proportional to CK activity
What are the different isoforms of CK?
CK-MB
CK-MM
CK-BB
What are the factors influencing rate of reaction?
Enzyme and substrate concentration
pH
Temperature
Inhibition
Activators/Co-enzymes
How does pH affect rate of reaction?
Max enzyme activity is at pH 7-8
Some enzymes are exceptions
ex. ALP functions at pH 10.5, pepsin functions at pH 1.5
What is the optimal temperature for an enzyme reaction?
37 C is optimal
How does inhibitors affect rate of reaction?
- Competitive inhibition
Analogue substrate competes for enzyme binding sites hence, reduces formation of desired product
ex. AST - Non-competitive inhibition
- Product inhibition
Product acts as an inhibitor of forward reaction
ex. ALP and inorganic phosphate
How does activators/coenzymes affect rate of reaction?
Molecules that increase the rate of reaction by promotion of the most active state of an enzyme or substrate
Metal ions can contribute to the tertiary structure of an enzyme for function
ex. ALP
- Zn = structure
- Mg = activator
How do we measure enzyme activity? Explain the difference between the 2 methods.
- Fixed time method
Measures the amount of change produced by an enzyme after the reaction reaches a fixed time interval - Continuous-monitoring method
Reaction is continuously monitored throughout the time interval
Why is the continuous-monitoring method better than the fixed time method for measuring enzyme activity?
- Fixed time method requires optimal selection of reaction conditions in order to lengthen linear phase and eliminate lag phases
- Continuous-monitoring method has a higher measurable upper limit and fewer samples require runs
What are aminotransferases? What are the two types?
Intracellular enzymes that catalyze amino acids’ reversible conversion to 2-oxo-acids
AST and ALT
Where is AST and ALT produced?
AST is produced heart, liver and skeletal muscle
ALT is produced in the liver and kidney
What are the differences between the reactions of AST and ALT?
Both use 2-oxoglutarate to form L-glutamate.
The difference is that AST uses L-aspartate and produces oxoloacetate while ALT uses L-alanine and produces pyruvate.
What are the clinical applications of AST and ALT?
Monitor liver disease
Why is ALT sufficient to monitor liver disease?
- ALT often higher than AST
- ALT is more tissue specific
- AST can be elevated in non-liver injury
Which method do we use to measure AST and ALT? What are the differences between the assays measuring AST and ALT?
Continuous monitoring method
For AST, oxoloacetate forms L-malate with the help of malate dehydrogenase
For ALT, pyruvate forms L-lactate with the help of lactase dehydrogenase
What are pre analytical considerations for AST and ALT?
AST:
Sample stability < 48 hrs
More susceptible to hemolysis than ALT
ALT:
Sample stability < 24 hrs
What are post analytical considerations for AST and ALT
Reference intervals:
AST is higher in neonates but no sex differences. ALT can be slightly different in neonates and also higher in males.
What is gamma glutamyltransferase?
Peptidase catalyzing peptide cleavage to smaller peptides or aa
It transfers a glutamyl group onto acceptor peptide.
Where is gamma glutamyltransferase (GGT) found?
Renal tubule
Pancreas
Liver
Intestine
In the microsomes of the cytoplasm and in the cell membrane
What is circulatory GGT an indicator of?
Hepatobiliary dysfunction
Circulatory GGT is from the hepatobiliary system.
How to test for GGT?
Donor: L-gamma-glutamyl-p-nitroanalide
Product: p-nitroanalide
Gives a yellow colour (405 nm)
What are some difficulties using L-gamma-glutamyl-p-nitroanalide as the donor for testing for GGT? What can we use instead?
Not water soluble at high concentrations
L-gamma-glutamyl-3-carboxy-4-nitroanilide
Read at 410 nm
Where can alkaline phosphatase (ALP) be found?
Small intestine
Proximal tubules (PT) of kidney
Liver (Measured ALP predominantly comes from liver)
Bone
Placenta
What are some of the clinical indications of ALP?
- Hepatobiliary disease
- Biliary obstruction triggers synthesis of ALP by hepatocytes which increases circulatory ALP
- Extrahepatic obstruction has 3x greater levels of ALP than intrahepatic
- Inflammation - Bone health
- Hyphophosphatasia: decreased ALP due to genetic inability for production, hence deficiency and poor bone growth
- Paget disease and bone cancer: increased ALP activity
- Bone fracture
How do we test for total ALP?
Chromogenic reaction
Activator: 2-amino-2-methyl-1-propanol (AMP)
How do we test for bone ALP?
- Immunoassays
- Densitometry
Use neuraminidase to remove sialic residues on bone ALP which can be visualized using densitometry; bALP peak is on the right, liver ALP is on the left
