Enzymes

Question 1

How are enzymes measured?

Answer 1

By the production or disappearance of a measurable substance. We don’t measure the enzyme itself but the activity of the enzyme.

Question 2

What are the 3 most common measurable substances used to measure enzymes?

Answer 2

Para-nitrophenol (yellow)
NADH, NADPH
H2O2 (peroxidase)

Question 3

How are coupled reactions used to measure enzymes?

Answer 3

The enzyme of interest is in reaction 1 known as the assay reaction. The product of reaction one is used as the substrate for reaction 2. The indicator reaction uses or produces something easy to measure.

Question 4

What is substrate depletion?

Answer 4

When you don’t have enough substrate to saturate all of the enzyme.

Question 5

What is product inhibition?

Answer 5

When you have too much product in the way which in turn inhibits the reaction.

Question 6

What is the Michaelis Menten constant?

Answer 6

The substrate concentration where the reaction velocity is half of the maximum.

Question 7

What is zero order kinetics?

Answer 7

When the maximum velocity has been reached and it doesn’t matter how much substrate you add this is as fast as it is going to go because the enzyme is saturated. The rate is independent of the substrate concentration.

Question 8

What is first order kinetics?

Answer 8

There is something limiting the reaction rate, if we increase the substrate concentration the rate will increase. The rate depends on substrate concentration.

Question 9

What is the most common enzyme kinetics shape to see in the lab?

Answer 9

Sigmoid as there is a lag phase at the start and it drops off at the end as you get product inhibition or substrate depletion.

Question 10

Why do you get a lag phase?

Answer 10

Because we use coupled reactions, this means there has to be enough product produced from the first reaction in order to become the substrate for the second reaction and get a measurable product.

Question 11

What are the 2 measuring systems used for quantifying enzymes?

Answer 11

International unit

Katal

Question 12

Which unit for quantifying enzymes is recommended?

Answer 12

The katal as it is an SI unit.

Question 13

What is the function of creatine kinase?

Answer 13

It catalyses the reversible phosphorylation of creatine by ATP this is dependent on Mg and inhibited by excess or other metal ions.

Question 14

Where is creatine kinase found?

Answer 14

Mostly found in skeletal muscle then heart, brain, GI, and urinary bladder.

Question 15

When would you see elevated creatine kinase?

Answer 15

In muscle damage such as myocardial infarction, muscular dystrophy, crush injuries and myocarditis.

Question 16

Why can’t creatine kinase be measured from an EDTA tube?

Answer 16

Because EDTA chelates magnesium which is required as a cofactor so creatine kinase activity will be close to 0.

Question 17

How is creatine kinase measured?

Answer 17

The reverse reaction from phosphocreatine to creatine, it is a coupled reaction with NADPH being measured.

Question 18

What is the most useful CK isoenzyme?

Answer 18

CKMB which is heart function specific therefore it can be used in suspected MI. It starts to rise 4 hours after the MI and remains until day 3 following. Usually measured by sandwich assay.

Question 19

What is the function of aminotransferases?

Answer 19

They transfer amino groups between amino acids for conversion into alpha keto acids and back.

Question 20

Where is AST found?

Answer 20

In the heart, liver, skeletal muscle and kidney. This is called a ubiquitous enzyme. Increased in haemolysis as found in RBCs.

Question 21

Why is AST measured?

Answer 21

It is useful in the assessment of hepatocellular disorders, also elevated in skeletal muscle damage, pulmonary embolism and muscular dystrophy.

Question 22

How do AST levels change after MI?

Answer 22

They are raised within 6-8 hours peak at 24 hours and normal within 5 days.

Question 23

How is AST measured?

Answer 23

Coupled reaction where oxoglutarate goes to oxaloacetate which goes to NAD which is measured.

Question 24

Where is ALT found?

Answer 24

It is most specific to the liver but also found in the kidney.

Question 25

Why is ALT measured?

Answer 25

It is useful in assessing hepatic disorders but the level depends on the degree and type of tissue damage. It is normal MI unless liver damaged also.

Question 26

How is ALT measured?

Answer 26

Rate of NADH decrease is measured.

Question 27

What is ALP?

Answer 27

A hydrolase that dephosphorylates various molecules at alkaline pH it is activated by divalent cations, needs Zn for reactions to occur. Inhibited by anions such as phosphate, borate, oxalate and cyanide.

Question 28

Where is ALP found?

Answer 28

It is ubiquitous but predominantly found in SI, kidney, bone, liver and placenta.

Question 29

When is ALP raised?

Answer 29

Usually from bone or liver such as hepatobiliary disease, bone disease or physiological states such as pregnancy, growth or healing.

Question 30

What is the function of GGT?

Answer 30

It transfers a gamma-glutamyl functional group from peptides to an acceptor, this is critical for maintaining levels of reduced glutathione.

Question 31

Where is GGT found?

Answer 31

Kidney, liver, pancreas, intestine.

Question 32

When is GGT raised?

Answer 32

Hepatobiliary disease such as cholestasis, cancers, hepatitis, alcoholism, obesity, drugs and pancreatitis.

Question 33

What is the function of lactate dehydrogenase?

Answer 33

It catalyses the oxidation of lactate to pyruvate.

Question 34

Where is LDH found?

Answer 34

Liver, heart, kidney, skeletal muscle, and red blood cells.

Question 35

When is LDH raised?

Answer 35

MI and muscle disorders
liver disorders
Haemolysis
Some cancers and tumour lysis syndrome.

Question 36

What is the function of amylase?

Answer 36

It catalyses the degradation of polymeric carbohydrates. It is very small and appears in the urine even if normal renal function.

Question 37

When is amylase raised?

Answer 37

Acute pancreatitis and salivary gland inflammation.

Question 38

When is lipase raised?

Answer 38

In acute pancreatitis if greater than 3x ULN it is very sensitive and specific. Peaks at 24 hours and decreased within 7-14 days.

Question 39

Why is lipase measured instead of amylase?

Answer 39

It is more specific, stable and persistent than amylase.

Question 40

How is lipase measured?

Answer 40

Spectrophotometrically but also by turbidimetry, titration, fluorometry, and immunoassay.