FHMP 012 enzymes in medicine

Question 1

what are the differences in fast and slow enzyme regulation

Answer 1

slow =

• hours/days
• in response to hormones like steroid or long term adaptations
• transcription rate
• mRNA stability
• Translation rate
• Degradation

fast =

• seconds/minutes
• in response to hormones e.g. adrenaline or metabolites
• location
• allosteric control
• covalent modification

Question 2

What does turnover mean?

Answer 2

• the number of substrates that can be converted into product from binding with enzymes per second
• an enzyme with a high turnover will be effected quicker from regulation

Question 3

what are covalent modifications?

Answer 3

Includes

• hydrolysis of the peptide
• addition of a molecule to a side chain or to the N- or C-terminus of the protein.
• phosphorylation
• very quick effects

Question 4

What is the allosteric site?

Answer 4

a site on the enzyme other than the active site

Question 5

What is an isozyme?

Answer 5

Enzymes that catalyse the same reaction but have different chemical composition and different physical properties

Question 6

describe how the isozymes PKF-2/FBPase-2 works

Answer 6

• it is a bifunctional enzyme (different versions of the same enzyme that have different effects in different parts of the body, like lipoprotein lipase)
• one catalyses forward reaction and one catalyses backwards reaction
• different isoforms in the heart, liver, and lung, and each organ enzyme is activated by different things

reaction:
fructose <–> fructose 2-6-bisphosphate
- PFK-2 = phosphofructokinase-2 = forwards
- FBPase-2 = fructose bisphophatase-2 = backwards

Question 7

what are the 5 types of enzyme inhibition?

Answer 7

• irreversible
• reversible
• competitive
• uncompetitive
• non-competitive

Question 8

What is irreversible inhibition?

Answer 8

1. Something covalently bonds to active site.
2. Cannot be removed.
3. lowers the concentration of active enzyme, but the affinity is the same ( as only the few modified enzymes stop binding to the substrate, the rest of the enzymes are unaffected and affinity is the same)
4. so Vmax decreases but Km is unchanged
   A few bind non-covalently.

Question 9

give an example of an irreversible inhibitor

Answer 9

penicillin,
aspirin:
- irreversibly binds inhibiting cyclooxgenase 1 and 2 which converts arachidonic acid into prostaglandins are thromboxanes ( used in clotting and inflammation)
- so it is anti-inflammatory, anti-platelet and anti-clotting so it is used to lower risk of heart attacks and as a pain killer with effects of blood thinning

Question 10

what is a reversible inhibitor?

Answer 10

• a substance that binds to an enzyme to inhibit it, but can be released
• so temporary decrease in Vmax when bound, but Km is not effected

Question 11

give an example of a reversible inhibitor

Answer 11

protease inhibitors

Question 12

What is competitive inhibition?

Answer 12

1. Compete for active site or bind to allosteric site and alter active site
2. Can be overcome by increasing substrate concentration.
3. increases the Km (more substrate required to achieve the same rate), but doesn’t change Vmax as the activity of enzyme isn’t affected
4. Often resembles the substrate/ similar structure

Question 13

give an example of a competitive inhibitor

Answer 13

• statins = used to lower cholesterol levels in blood
• class of HMG-CoA reductase which is the rate-limiting enzyme in cholesterol bio-synthesis
• statins inhibit HMG-CoA and cause accumulation of mevalonate (substrate) which signals to decrease cholesterol metabolism and uptake it from the blood

Question 14

what is an uncompetitive inhibitor?

Answer 14

1. binds only to the enzyme-substrate complex (cannot bind to enzyme alone) -think of protective UNcle = only around when get a bf (a substrate)
2. moves the E + S = ES = EP equilibrium towards ES which increases the affinity of the enzyme for the ones that aren’t inhibited but decreases the activity overall as some are inhibited
3. so the Km is decreased and the Vmax is decreased

Question 15

give an example of an uncompetitive inhibitor

Answer 15

• lithium used to treat manic depressive psychosis and bipolar disorder
• an uncompetitive inhibitor of inositol monophosphatase which is used in phosphoinositide signalling which affects brain activity, such as reducing dopamine and glutamate but increases GABA and serotonin
• a small therapeutic range so lithium levels need to be regularly measured to avoid toxicity

Question 16

What is non-competitive inhibition?

Answer 16

1. Doesn’t attach to the active site, attaches to the allosteric site, and changes the shape of the enzyme /active site so the substrate doesn’t fit as well.
2. can bind to the bound or unbound enzyme, but doesn’t resemble the substrate
3. Lowers Vmax, Km is unchanged ( as fewer enzymes can bind so activity decreases and the active site is altered to increasing the substrate will not make a difference)
4. binds to both E and ES so E + S = ES equilibrium is unchanged so Km is unchanges

Question 17

give an example of non-competitive inhibitors

Answer 17

• protein kinse inhibitors

- heavy metal ions

Question 18

what are protein kinase inhibitors?

Answer 18

• protein kinases are an important group of enzymes which modify proteins by phosphorylating them which usually activates them
• protein kinase inhibitors are usually ATP-competitive, but ATP conc in cells is very high so the competitive inhibitors need a very high affinity to have an effect and have low specificity
• so we need to find a non-competitive inhibitor which is more specific and is not affected by ATP

Question 19

why are enzymes used for diagnosis?

Answer 19

• because they are highly specific, they can be used to test for certain substances to verify presence or measure conc

Question 20

describe 5 ways enzymes are used in diagnosis

Answer 20

1. measurement of metabolites
2. glucose peroxidase enzyme (glucose conc)
3. measurement of enzymes
4. myocardial infarction (CK-MB detection)
5. liver disease ( measure enzyme conc associated with fatty liver disease)

Question 21

describe using enzymes to measure metabolites

Answer 21

• sample should be diluted so substrate conc < Km and enzyme conc should be in excess
• this is so the enzyme is not saturated
• so rate of reaction is dependent on conc of metabolite/substrate you want to measure

Question 22

describe using enzymes for glucose peroxidase assay

Answer 22

• alpha glucose reacts with mutarotase to form beta glucose which reacts with glucose oxidase to form hydrogen peroxide
• peroxidase uses hydrogen peroxide to oxidise a colourless compound and make it coloured
• so the absorption of this colour = glucose conc in sample

Question 23

describe the measurement of enzymes method

Answer 23

• enzyme should be saturated, so high substrate conc
• Vmax is recorded
  1 unit of enzyme activity = 1 micromole of substrate converted per minute

Question 24

describe how enzymes are used to detect myocardial infarction

Answer 24

• injury of heart tissue during myocardial infarction causes cellular proteins to be leaked into plasma
• CK = creatine kinase which is found in lots of tissues but only CK-MB is found in the heart but is normally trapped in cells
• so CK-MB presence in plasma can confirm a recent/current myocardial infarction

Question 25

describe how enzymes can be used to detect liver disease

Answer 25

• healthy livers should have little to no fat, but alcoholism and bad diet can cause accumulation of fat in the liver and liver disease
• liver disease causes hepatic enzymes to leak out into the plasma
• specifically AST (aspartate transaminase) and ALT (alanine transaminase) are characteristically found elevated in plasma of those with fatty liver disease
• AST:ALT > 1.5 = alcoholic fatty liver disease
• AST:ALT < 1 = non-alcoholic fatty liver disease