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IMS topic 1 > Enzymes > Flashcards

Enzymes Flashcards

1
Q

Why are many drugs enzyme inhibitors?

A

They mmic the shape and structure of th molecule that interacts with the enzyme to knock out the enzymes activity.

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2
Q

What is the equation for equilibrium constant?

A

K = [concentration of B]/[concentration of A]

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3
Q

How do enzymes work? (simply)

A

Increase the rate at which equilibrium is reached by decreasing the activation energy of the reaction

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4
Q

How do enzymes decrease the activation energy?

A

By providing catalytically competent groups for a specific reaction mechanism, binding substrates at an orientation that is optimised of the reaction, and stabilising transition states of the substrate (to stop the reaction from going backwards, and promote the forward reaction)

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5
Q

Define assay

A

a procedure for measuring the biochemical or immunological activity of proteins/enzymes in a sample

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6
Q

Define Km in terms of the Michaelis Menten model

A

Km is the substrate concentration that gives an initial reaction velocity to half the maximal rate (affinity of an enzyme for the substrate)

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7
Q

What does a high Km indicate?

A

A low affinity of the enzyme for the substrate (more substrate is needed to get to a particular velocity)

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8
Q

What does a low Km indicate?

A

A high affinity of the enzyme for the substrate

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9
Q

What is the equation for Km?

A

Km = (K2 + K3)/(K1)
Where K1 is the rate constant for the forward reaction of E + S –> ES; K2 is the rate constant of the backward reaction of ES –> E + S and K3 is the rate constant of the forward reaction of ES –> E + P

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10
Q

What is the michaelis mention equation for V (rate of formation of product)?

A 
V = Vmax ([S]/([S} + Km)) 
V = rate of formation of product
Vmax = theoretical maximum rate of reaction (will increase with more enzyme)
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11
Q

Does Km ever change within a particular enzyme?

A

no

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12
Q

What is Vmax?

A

The theoretical maximum rate of reaction (will never be reached) (all enzyme saturated with substrate)

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13
Q

How else can Km be defined? What needs to be drawn in a graph to calculate this?

A

Can be read off the graph at the X axis at Y axis Vmax/2
You cannot reach Vmax in the lab, so a double reciprocal of the equation is written in the form y = mx + c —> 1/v = Km/vmax[s] + 1/vmax and plotted as 1/rate by 1/substrate concentration

The X intercept is then the same as Km

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14
Q

Where in the body can enzymes work at lower temperatures?

A

Testicles

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15
Q

Why do enzymes not work at lower than optimum temperature?

A

The substrate won’t bind

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16
Q

How do IRREVERSIBLE inhibitors work? Give an example

A

COVALENT modification of amino acid side chains
e.g. Parathion damages enzymes in the nervous system by blocking acetylcholinestarse which breaks down acetylcholine in nervous system pain pathways to switch off impulses. When blocked, impulses keeps going and drive muscle movements, causing spasm and suffocation if it reaches the lungs.

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17
Q

How does competitive inhibition work?

A

The enzyme can bind to the substrate of the inhibitor, but not both; the substrate an inhibitor share similar structures. There is completion for the active site, meaning inhibition can be overcome by high substrate concentration.

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18
Q

How are Vmax and Km affected by competitive inhibition?

A 
Vmax unchanged
Km increased (so lower affinity for substrate)
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19
Q

How is a new drug defined as competitive or non competitive

A

Whether or not it changes the Km value

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20
Q

How does a non-competitive inhibitor work?

A

The inhibitor and the substrate can bind to the enzyme simultaneously - the binding occurs at different sites.
The inhibitor alters the conformation of the active site
inhibition is not affected by substrate conc.

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21
Q

How are Vmax and Km affected by non-comepetive inhibition?

A

The Vmax decreases (less substrate can bind)

Km is unchanged (substrate binding to uninhibited enzyme is unchanged)

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22
Q

What is the IC50 value an inhibitor?

A

Measures the effectiveness of the inhibitor - the inhibitory concentration that knocks out 50% of the ability of the enzyme

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23
Q

What is an NSAID?

A

Non-steroidal anti-inflammatory drugs

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24
Q

How do NSAIDs work?

A

Block COX-2 enzyme, which synthesises prostoglandins that cause inflammation, pain and fever.
However, they also inhibit COX-1 which is an enzyme essential for health of the stomach and kidney

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25
Q

How do NSAIDs work in aspirin?

A

Aspirin covalently modifies a SERINE residue in the active site. Inhibitor binding is competitive and irreversible.

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26
Q

How do NSAIDs work in ibuprofen?

A

Binds to the active site, not covalent to reversible. Competitive.

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27
Q

Why are metal ions important cofactors for enzyme function and how do they enter the body?

A

Metal ions are provided as trace elements in the diet, they are essential components in the active sites of enzymes. Some ions occur naturally.
They bind electrostatically to the active site or act as oxidising agents.
Some metal ions are not part of the active site but are required for activity (e.g. Ca2+)

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28
Q

Why are co-enzymes important for enzyme function?

A

They are carriers of reaction components e.g. NADH and FADH2 carry electrons
Coenzyme A carries acyl units

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29
Q

What is the role of glucose-6-phosphate dehydrogenase (G6DPH) in the body? What are the symptoms of a deficiency?

A

It produces most of the body NADPH which is need for synthesis of nucleic acids, RNA synthesis, lipids, glycolysis etc. A deficiency can cause haemolytic crises triggered by certain foods/infections, jaundice, brain damage.

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30
Q

What decides the fate of G6DPH in terms of glycolysis or the pentose pathway?

A

The requirements of the cell - if there is rapid cell growth and high demand for RNA synthesis the pentose pathway is used, if there are high energy demands the glycolysis pathway is used.

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31
Q

What regulates G6DPH?

A

levels of oxidised NADP+; high levels of this signals a need for more NADPH

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32
Q

Why do we need NADPH?

A

It maintains a strong reducing environment in the cell by maintaining levels of glutathionine reductase which in its reduced state MOPS UP FREE RADICALS such as hydrogen peroxides which attack lipids.

33
Q

Why does a G6DPH deficiency particularly affect RBCs?

A

Most cells can make their own NADPH using the mitochondria in different pathways to maintain levels of glutathionine reductase and mop up free radicals, but RBCs do not have a mitochondria. Lipids in RBCs are destroyed by free radicals, causing haemolytic lysis.

34
Q

What can trigger heamolysis in people with a G6DPH deficiency?

A

Anti-malarial drug primaquine - it stimulates peroxide formation which are free radicals that attack lipids in RBCs, which do not have another pathway to make NADPH and maintain glutathionine reductase levels to mop up free radicals

35
Q

What are serine protease inhibitors (‘serpins’)?

A

Small natural proteins that inhibit enzyme activity very efficiently by their ability to fit precisely into the active site of many enzymes. they are non-specific.
e.g. antithrombin ||| shuts off the blood clotting system

36
Q

Why are serine protease inhibitors dangerous? How are they controlled in the body?

A

They are non-specific and very efficient so can cause damage in the wrong place at the wrong time. They have inhibitors that will stop their activity e.g. pancreatic trypsin inhibitor stops trypsin from digesting the pancreatic cells

37
Q

What is feedback regulation?

A

The enzyme may be regulated directly by the product of its own reaction to promote or inhibit the reaction. If this decreases activity, it forms a negative feedback loop

38
Q

Which proteins carry out protein phosphorylation to enzymes as a form of covalent modification, how and why?
Which protein removes phosphate groups?

A

Proteinkinases, this changes the active site to activate or inactivate the enzyme, controlling hormonal signalling.
Phosphate groups can be removed by phosphates, or y-phosphate can be transferred onto a residue.

39
Q

What is a zymogen?

A

An inactive pro-enzyme

40
Q

How are zymogens activated? Is this reversible?

A

proteolysis; part of the polypeptide chain is removed by proteolytic enzymes
Irreversible

41
Q

Why are proteolytic enzymes stored as inactive zymogen precursors?

A

They are destructive and could cause damage

42
Q

Why are antitrypsin and antithrombin found at high concentrations?

A

Tight control of proteolytic enzymes

43
Q

What is chromotrypsin?

A

A serine protease secreted by the pancreas (a digestive enzyme), which breaks down proteins by cleaving the peptide bonds. It has 3 polypeptide chains linked by disulphide bonds.

44
Q

How is chromotyrpsin made and activated?

A

Chymotrypsin (the zymogen) is synthesised in pancreas cells (acinar cells) and secreted into the duodenum (first section of the small intestine).
Trypsin cleaves chymotrypsin to form pi-chymotrypsin which is then cleaved again by already active a-chymotrypsin to make a-chymotrypsin. This process is self regulating,

45
Q

How is trypsin activated?

A

The zymogen trypsinogen is cleaved by a duodenal enzyme enteropeptidase, removing amino acids 1-6 to activate trypsin. This causes a small conformational change. Trypsin also switches on other gut enzymes.

46
Q

What is protein phosphorylation?

A

The addition of P from ATP

47
Q

Why are serpins so effective?

A

A ‘perfect fit’ to the active site

48
Q

How does the prothrombin zymogen activate the clotting cascade?

A

The prothrombin zymogen is bound to the external surface of platelets, linked to the negatively charged phospholipids of the membrane by Ca2+ ions. Prothrombrin is cleaved to thrombin from the platelet by FXa

49
Q

How does antithrombin ||| prevent blood clotting?

A

Binds tightly to thrombin and serine protease factors

50
Q

What is the key protein in clot lysis?

A

TPA

51
Q

What is the role of TPA (tissue-type plasminogen activator) in clot lysis?

A

TPA adheres to the clot and binds to plasminogen (inactive zymogen precursor plasmin) to target activity to the right place. TPA activates plasminogen to plasmin by serine protease activity, and plasmin digests the blood clot to small peptides.

52
Q

How is recombinant TPA used as a drug?

A

Injected into patients to remove blood clot and restore blood supply in coronary thrombosis. (known as clot busters)

53
Q

Why are enzymes in the blood an indicator of specific damaged tissue?

A

Most enzymes will not normally be in the blood, and should only be found in specific tissues

54
Q

Why do not all enzymes in the blood point to specific tissue damage?

A

some enzymes are vital for metabolic processes in all cells

55
Q

What does the release of mitochondrial enzymes into the blood indicate?

A

Severe damage to cells

56
Q

Why does time after injury matter when measuring enzyme levels?

A

Different enzymes have different half lives

57
Q

What is 1IU?

A

The amount of energy that will convert 1 micromole of substrate per minute under standard defined conditions

58
Q

In LFTs, which protein at low levels is a broad indicator of an unhealthy liver?

A

Albumin (should be <3.5-5.3IU)

59
Q

What do GOT and GPT show in LFTs and why are they used as a ratio?

A 
High GOT (should be 6-48IU) suggests acute liver damage, but could be from RBCs or muscles. High GPT (should be 7-55IU) is a more specific indicator of liver damage; high activities rule out muscle damage. 
Total transaminase (GOT/GPT); if both levels are high but with a normal ALP this suggests liver cell death, e.g. cancer, alcohol, virus. If levels are over 1000IU then could be paracetamol poisoning or severe liver failure. The ratio discriminates between muscle and liver damage.
60
Q

What do elevated levels of Alkaline Phosphatase (ALP, removes phosphate groups from metabolites) and GGT (gamma-glutamyl transferase) show?

A

Levels should be 45-120IU/L

Elevated levels indicate bile duct obstruction (but can be high in growing kids & pregnancy) `

61
Q

What is jaundice?

A

A build up of bilirubin in the blood caused by a failure of the liver to breakdown the waste product of RBC breakdown

62
Q

Why does a slow prothrombin time indicate liver failure?

A

Failure of the liver to synthesise and secret blood clotting proteins such as prothrombin. Also used to monitor warfarin usage and vitamin K status

63
Q

Give three properties needed for an enzyme to be a good diagnostic marker

A

Found in high concentrations in specific tissues
Limited in/absent from other tissues
Changes in serum level are linked to only one pathology
Relatively narrow range of activities
Long diagnostic window of opportunity (can be detected in the blood for a while)
Easy to assay (reliability, sensitivity)
Direct correlation with severity of symptoms

64
Q

Define isoenzyme, why are these important as diagnostic markers?

A

Has similar forms that perform specialised enzymatic reactions in a particular tissue. Catalyse the same reaction but have different substrate affinity and different sensitivity to inhibitors. (have a different Km).
They have different tissue specific distribution, so if you can identify which isoenzyme is present you can identify the site of injury

65
Q

Levels of which four enzymes are measured after cardiac injury?

A

Creatine Kinase
Myoglobin
Cardiac Troponin
Lactate dehydrogenase

66
Q

What is the role of creatine kinase (CK)?

A

It is the enzyme in the reaction of creatine and ATP to form creatine phosphate + ADP + H+
Creatine phosphate is a store of ‘high energy’ phosphate groups in muscles
When ATP levels are low, the reaction reverses to make more ATP

67
Q

How do levels of CK in the blood point to heart damage?

A

CK is made of two polypeptide, an M and a B subunit. MM is found in skeletal muscle and MB is found mainly in the myocardium, so levels increase in acute myocardial infarction (AMI). BB is found in the brain.

68
Q

Why are levels of CK and other enzymes still monitored a while after heart damage?

A

The speed at which they drop can be an indicator of how susceptible the patient is to another AMI

69
Q

What is the structure of lactose dehydrogenate and where are the two forms normally found? What changes after AMI?

A

4 subunits, made from combinations of two polypeptides, M and H. H is abundant in blood cells and the heart and M is abundant in muscles, kidney and liver.

LDH1 (H4) has a higher affinity for substrate and is mainly found in the heart to make pyruvate from lactate. LDH2 is normally present in serum. After AMI LDH1 levels in serum rise; peaks at 2-3 days.

70
Q

What does high LDH3 (lactose dehydrogenate hormone) indicate?

A

Pulmonary disease

71
Q

Other than AMI, what else can a flipped LDH1:LDH2 indicate?

A

Anaemia, cancer, angina, haemolysis, can be present in highly trained athletes

72
Q

What drug is used in the treatment of paracetamol poisoning? Why is a precise dose required? How is it measured?

A

N-acetylcysteine/methionine
This can have toxic affects
Paracetamol levels are measured using a bacterial enzyme which converts the drug to amino phenol. This is reacted with oath-cresel in the blood in the presence of copper ions to make a coloured dye. the colour is quantified.

73
Q

Why is excess paracetamol a problem? How does acetylcysteine/methionine combat this?

A

Paracetamol is normally metabolised and tagged for secretion by the kidneys. At high doses, the enzyme cytochrome p450 metabolises paracetamol to a highly reactive quinoe compound, which reacts with glutathionine in the cytoplasm to render it harmless.
When this reaction is swamped, the quinces attack proteins and lipid membranes of liver cells, compromising liver functions.
Acetylcysteine boosts the amount of available glutathionine to increase the body’s capacity to get rid of harmful quinoes.

74
Q

How is blood glucose monitored by patients themselves?

A

Simple biochemical assays using a glucose specific enzyme that generates hydrogen peroxide. A second enzyme reacts this with a dye molecule, changing colour when oxidised by the peroxide activity. Both enzymes are immobilised and placed onto a plastic strip which is placed in a reader, which gives a reading of metabolite concentration.

75
Q

How are enzyme electrodes used as immobilised enzymes in estimating the concentrations of various metabolites? e.g. blood alcohol, urine pH, cholesterol
Give two advantages of electrodes

A

The enzyme is trapped in a permeable membrane in the electrode. The analyte permeates through the membrane, coming into contact with the enzyme. If the reaction converts the analyte to an ionic species, the electrical conductivity of thhe solution will change in proportion to the concentration of the ion = you can measure the electrical signal of the original conc. of the analyte. A transducer converts this to a numerical form.

This is fast
Can be done in large quantities in labs

76
Q

How can enzymes be used in cancer therapy?

A

Asparagine is an amino acid required for ammonia synthesis (so synthesis of amino acids), and is normally made by the residues aspartate and glutamine. Tumour cells lack the enzyme asparagine synthesise so cannot make asparagine. This can be excluded from the diet and some patients of CALL (childhood acute lymphoblastic leukaemia) are treated with an injection containing the enzyme asparaginase that destroys circling asparagine. Tumour cells cannot make proteins and grow.
Normal cells are unaffected.

77
Q

How are enzymes used in kidney dialysis?

A

A patients blood is circulated though a cell with a semi-permeable membrane with urease on the opposite side. A high conc of urea in the blood causes it to diffuse across the membrane, where it is converted to CO2 and NH3. Ammonia is absorbed into charcoal. CO2 permeates back across the membrane where it absorbed in the blood and is necessary to maintain pH.

78
Q

Define enzymopathy

A

Genetic defects causing loss of enzyme activity; deficiency, absence or inactivation of a single enzyme

79
Q

How can gene therapy help cure enzymopathies?

A

Cloning of a new gene allows production of a protein using recombinant DNA technology

IMS topic 1 (7 decks)

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