Lecture 1: Enzymology Flashcards by Rachel Winardi

What are enzymes?

Proteins with catalytic properties

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What are the 6 different classes of enzymes?

Oxidoreductase
Transferase
Hydrolase
Lyase
Isomerase
Ligase

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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

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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

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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

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What are isoenzymes?

Enzymes that are related but differ based on aa sequence

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

Physical and biochemical properties
Immunologic properties
Physiological role

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What are the clinical relevant isoenzymes?

LDH, CK, Amylase and ALP

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What type of lab tests are used to assess isoenzymes?

Electrophoresis - size and charge differences
Chromatography - size, charge and polarity
Immunoassay - aa sequence

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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

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What are some cons of testing for macroenzymes?

Longer clearance, and significant elevation in plasma
Clinically benign - not indicative of injury/disease

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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

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What is the purpose of the Michaelis-Menten Curve?

Assess velocity at varying substrate concentrations
Determine Km
Identify possible inhibition at high concentration

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What is the Lineweaver-Burk Transformation?

Inverse transformation of velocity & substrate concentration

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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

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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

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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?

1. ALT often higher than AST 2. ALT is more tissue specific 3. 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?

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

How do we test for total ALP?

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

How do we test for bone ALP?

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