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PATH 406 > Lecture 1: Enzymology > Flashcards

Lecture 1: Enzymology Flashcards

1
Q

What are enzymes?

A

Proteins with catalytic properties

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2
Q

What are the 6 different classes of enzymes?

A

Oxidoreductase
Transferase
Hydrolase
Lyase
Isomerase
Ligase

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3
Q

What is enzymology used for in the clinical scene?

A
  1. Application of enzyme analysis in diagnosis or treatment of disease
  2. Determine enzyme deficiency
  3. Indicate tissue damage
  4. Indicate organ failure
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4
Q

What are some enzymes used to determine liver function, pancreas function, and general cell damage?

A

Liver function: AST, ALT, ALP, GGT
Pancreas: Amylase, Lipase
General Cell Damage: LDH

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5
Q

What is the protein structure of an enzyme and what does it need to function properly?

A

3D structure required to be functional
Multiple 3D structures may be required for proper enzyme activity

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6
Q

What are isoenzymes?

A

Enzymes that are related but differ based on aa sequence

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7
Q

What properties change due to the different aa sequences in isoenzymes?

A
  1. Physical and biochemical properties
  2. Immunologic properties
  3. Physiological role
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8
Q

What are the clinical relevant isoenzymes?

A

LDH, CK, Amylase and ALP

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9
Q

What type of lab tests are used to assess isoenzymes?

A
  1. Electrophoresis - size and charge differences
  2. Chromatography - size, charge and polarity
  3. Immunoassay - aa sequence
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10
Q

What are macroenzymes? What are some examples?

A

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

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11
Q

What are some cons of testing for macroenzymes?

A
  1. Longer clearance, and significant elevation in plasma
  2. Clinically benign - not indicative of injury/disease
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12
Q

How do we express enzyme activity?

A

U/L = unit of activity/volume
where U is the quantity of enzyme that catalyzes 1 umol substrate/min

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13
Q

What is the purpose of the Michaelis-Menten Curve?

A
  1. Assess velocity at varying substrate concentrations
  2. Determine Km
  3. Identify possible inhibition at high concentration
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14
Q

What is the Lineweaver-Burk Transformation?

A

Inverse transformation of velocity & substrate concentration

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15
Q

Differentiate between a 0 order and 1st order reaction.

A

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

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16
Q

How does the creatine kinase assay work? Which reactions are 0 order and 1st order? How do you know?

A

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

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17
Q

What are the different isoforms of CK?

A

CK-MB
CK-MM
CK-BB

18
Q

What are the factors influencing rate of reaction?

A

Enzyme and substrate concentration
pH
Temperature
Inhibition
Activators/Co-enzymes

19
Q

How does pH affect rate of reaction?

A

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

20
Q

What is the optimal temperature for an enzyme reaction?

A

37 C is optimal

21
Q

How does inhibitors affect rate of reaction?

A
  1. Competitive inhibition
    Analogue substrate competes for enzyme binding sites hence, reduces formation of desired product
    ex. AST
  2. Non-competitive inhibition
  3. Product inhibition
    Product acts as an inhibitor of forward reaction
    ex. ALP and inorganic phosphate
22
Q

How does activators/coenzymes affect rate of reaction?

A

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

23
Q

How do we measure enzyme activity? Explain the difference between the 2 methods.

A
  1. Fixed time method
    Measures the amount of change produced by an enzyme after the reaction reaches a fixed time interval
  2. Continuous-monitoring method
    Reaction is continuously monitored throughout the time interval
24
Q

Why is the continuous-monitoring method better than the fixed time method for measuring enzyme activity?

A
  1. Fixed time method requires optimal selection of reaction conditions in order to lengthen linear phase and eliminate lag phases
  2. Continuous-monitoring method has a higher measurable upper limit and fewer samples require runs
25
Q

What are aminotransferases? What are the two types?

A

Intracellular enzymes that catalyze amino acids’ reversible conversion to 2-oxo-acids

AST and ALT

26
Q

Where is AST and ALT produced?

A

AST is produced heart, liver and skeletal muscle
ALT is produced in the liver and kidney

27
Q

What are the differences between the reactions of AST and ALT?

A

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.

28
Q

What are the clinical applications of AST and ALT?

A

Monitor liver disease

29
Q

Why is ALT sufficient to monitor liver disease?

A
  1. ALT often higher than AST
  2. ALT is more tissue specific
  3. AST can be elevated in non-liver injury
30
Q

Which method do we use to measure AST and ALT? What are the differences between the assays measuring AST and ALT?

A

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

31
Q

What are pre analytical considerations for AST and ALT?

A

AST:
Sample stability < 48 hrs
More susceptible to hemolysis than ALT

ALT:
Sample stability < 24 hrs

32
Q

What are post analytical considerations for AST and ALT

A

Reference intervals:
AST is higher in neonates but no sex differences. ALT can be slightly different in neonates and also higher in males.

33
Q

What is gamma glutamyltransferase?

A

Peptidase catalyzing peptide cleavage to smaller peptides or aa

It transfers a glutamyl group onto acceptor peptide.

34
Q

Where is gamma glutamyltransferase (GGT) found?

A

Renal tubule
Pancreas
Liver
Intestine

In the microsomes of the cytoplasm and in the cell membrane

35
Q

What is circulatory GGT an indicator of?

A

Hepatobiliary dysfunction

Circulatory GGT is from the hepatobiliary system.

36
Q

How to test for GGT?

A

Donor: L-gamma-glutamyl-p-nitroanalide
Product: p-nitroanalide
Gives a yellow colour (405 nm)

37
Q

What are some difficulties using L-gamma-glutamyl-p-nitroanalide as the donor for testing for GGT? What can we use instead?

A

Not water soluble at high concentrations
L-gamma-glutamyl-3-carboxy-4-nitroanilide
Read at 410 nm

38
Q

Where can alkaline phosphatase (ALP) be found?

A

Small intestine
Proximal tubules (PT) of kidney
Liver (Measured ALP predominantly comes from liver)
Bone
Placenta

39
Q

What are some of the clinical indications of ALP?

A
  1. 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
  2. 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
40
Q

How do we test for total ALP?

A

Chromogenic reaction
Activator: 2-amino-2-methyl-1-propanol (AMP)

41
Q

How do we test for bone ALP?

A
  1. Immunoassays
  2. 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

PATH 406 (24 decks)

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