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enzymes Flashcards

1
Q

how do most drugs work?

A

they are enzyme inhibitors

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2
Q

what is the relevance of enzymology to medicine?

A

drug action
defects in enzymes underlie disease
clinical diagnosis and prognosis

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3
Q

what is an enzyme?

A

biological catalyst of chemical reactions specific to a reaction that is more often than not a protein

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4
Q

what don’t enzymes do?

A

do not shift the place of equilibrium

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5
Q

how do enzymes decrease the activation energy?

A

By providing catalytically competent groups for a specific
reaction mechanism.
By binding substrates such that their orientation is optimised for the reaction
By preferentially binding and stabilising transition
states of the substrate.

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6
Q

what is an active sight?

A

is the region of the enzyme at which substrate binding and conversion to product takes place.
is a 3-dimensional entity comprising crucial amino acid residues.

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7
Q

how does the active site bind to substrates?

A

binds substrate via multiple weak interactions

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8
Q

how does an active site provide specificity?

A

provides specificity because of its unique conformation of atoms

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9
Q

how does an active site provide specificity?

A

provides specificity because of its unique conformation of atoms

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10
Q

what is an assay?

A

A procedure for measuring the biochemical or immunological activity of a sample

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11
Q

how do we measure enzyme activity?

A

the Michaelis-Menten model

E+S ES –> E+P

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12
Q

what is K1?

A

the rate of formation of enzyme-substrate complex

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13
Q

what are K2 + K3?

A

the rates of dissociation of ES complex

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14
Q

how do you work out the michaelis constant?

A

(K2 + K3) / K1

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15
Q

what is the michaelis-menton equation?

A

rate of formation (v) = Vmax times substrate concentration [S] over substrate concentration + michaelis constant (Km)

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16
Q

what is the significance of the michaelis menton equation?

A

Km is the substrate concentration at which the reaction
rate is half its theoretical maximum value.
V= Vmax/2

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17
Q

how is Vmax derived experimentally?

A

Lineweaver-Burk double reciprocal plots

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18
Q

what does the x-intercept show on a Lineweaver-Burk plot?

A

-1/Km

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19
Q

what does the y-intercept show on a Lineweaver-Burk plot?

A

1/Vmax

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20
Q

what does the gradient show on a Lineweaver-Burk plot?

A

Km/Vmax

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21
Q

what factors affect enzyme catalysed reactions?

A

Substrate (or enzyme) concentration
Temperature
pH
Inhibitors- both natural and exogenous

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22
Q

what is irreversible inhibition?

A

Covalent modification of amino acid side chains in the active site

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23
Q

what are the two forms of reversible inhibition?

A

Competitive and non-competitive

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24
Q

what does the gradient show on a Lineweaver-Burk plot?

A

Km/Vmax

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25
Q

what are the two forms of reversible inhibition?

A

Competitive and non-competitive

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26
Q

what is competitive inhibition?

A

Substrate and inhibitor compete for active site

Substrate and inhibitor often share similar structures

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27
Q

how does competitive inhibition affect a Lineweaver-Burk plot?

A

Vmax unchanged - can be overcome by high substrate conc
Km increased - steeper gradient
Higher [S] needed to reach rate Vmax/2.

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28
Q

what is non-competitive inhibition?

A

Inhibitor and substrate can bind simultaneously.
Binding occurs at independent sites.
Inhibitor alters conformation or accessibility of active site.
Inhibition not affected by high substrate concentration

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29
Q

Effects of non-competitive inhibition on the Lineweaver-Burk plot

A 
Vmax decreased (proportion of enzyme ‘switched off’)
 Km unchanged (substrate binding to uninhibited enzyme unaffected)
 Increasing substrate concentration has no effect
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30
Q

what is an IC50 value?

A

the concentration at which activity is at 50%

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31
Q

how does aspirin inhibit COX-1?

A

Covalent modification of a serine residue in the active site

Inhibitor binding is competitive, inhibition is irreversible

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32
Q

how does ibuprofen inhibit COX-1?

A

Binds to active site, but not covalently attached

Inhibitor binding is competitive, inhibition is reversible

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33
Q

how does ibuprofen inhibit COX-1?

A

Binds to active site, but not covalently attached

Inhibitor binding is competitive, inhibition is reversible

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34
Q

why are metal ions important?

A

cofactor for enzyme function

Metal ion may bind reactants electrostatically, or may act as oxidising agents.

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35
Q

why are coenzymes important?

A

function as carriers of reaction components

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36
Q

how do NADH and NADPH differ?

A

NADPH carries an additional phosphate group on the ribose moiety of the adenosine nucleoside component

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37
Q

what is NAD+ a cofactor of?

A

conversion of ethanol to acetaldehyde by the enzyme alchohol dehydrogenase

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38
Q

what is biotin a cofactor of?

A

Acetaldehyde can be metabolised to pyruvate by pyruvate carboxylase

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39
Q

what is biotin a cofactor of?

A

Acetaldehyde can be metabolised to pyruvate by pyruvate carboxylase

40
Q

how does coenzyme A work?

A

same base-ribose-phosphate ‘handle’ as NADH and NADPH.
The active part of the molecule is the thiol-containing pantothenic acid that is used to shuttle carbon units in metabolic reactions.

41
Q

what does glucose-6-phosphate dehydrogenase deficiency cause?

A

restricted ability to make NADPH in particular

can trigger a haemolytic crisis and anaemia in some carriers of G6PDH mutations

42
Q

what does the pentose phosphate pathway produce?

A

Ribose 5-phosphate, NADPH

43
Q

when is the PPP selected for?

A

rapid cell growth or a high demand for RNA synthesis

44
Q

what is the committed step in the PPP?

A

glucose 6-phosphate dehydrogenase senses the NADP+/NADPH levels committing glucose 6-phosphate to the pentose phosphate pathway in order to increase production of NADPH

45
Q

what does Genetically inherited deficiency in G6PDH induce?

A

Primaquine-induced haemolytic anaemia

46
Q

why else is NADPH important?

A

NADPH is also very important in maintaining the levels of reduced glutathione in cells. this reduces lipid hydroperoxides to alcohols in a reaction catalysed by glutathione peroxidase

47
Q

what are serpins?

A

Serine protease inhibitors
small proteins produced naturally in the body that block enzyme activity. extremely good fit to the active site of the protease

48
Q

what is the universal mechanism of enzyme modulation?

A

Protein phosphorylation

49
Q

what controls the phosphorylation of enzymes?

A

protein kinases

50
Q

how are phosphate groups removed?

A

phosphatases

51
Q

what is a zymogen?

A

a pro-enzyme

52
Q

how are zymogens activated?

A

proteolysis

53
Q

why are enzymes stored as zymogens?

A

their destructive potential

54
Q

what is chymotrypsin

A

digestive enzyme secreted by the pancreas into the duodenum in an inactive form

55
Q

what is chymotrypsin?

A

digestive enzyme secreted by the pancreas into the duodenum in an inactive form

56
Q

what is the catalytic triad?

A

Serine, Histidine and Aspartate

57
Q

which enzymes are chymotrypsin related to?

A

trypsin and elastase

58
Q

where does trypsin cleave?

A

cleaves bonds C-terminal to lysine and arginine

59
Q

where does elastase cleave?

A

cleaves C-terminal to glycine and alanine

60
Q

where does chymotrypsin cleave?

A

cleavage C-terminal to hydrophobic residues

61
Q

where is chymotripsinogen synthesised?

A

synthesised in acinar cells of pancreas

62
Q

how is chymotrypsinogen activated?

A

Trypsin cleaves the inactive chymotrypsinogen close to its N-terminal end
Active chymotrypsin already in the duodenum cuts at a second site, giving the fully active α-chymotrypsin

63
Q

how is trypsinogen activated?

A

cleavage of a small N-terminal peptide switches on the enzyme by active trypsin

64
Q

what are the 5 levels enzymes can be controlled?

A
  1. Inhibition (reversible or irreversible)
  2. Feedback regulation
  3. Covalent modification
  4. Proteolytic activation
  5. Regulation of protein synthesis/breakdown.
65
Q

what is the central role of serine proteases?

A

blood clotting cascade

66
Q

what initiates the clotting cascade?

A

the enzymes kinin and kalikrein released by damaged tissue initiate the process

67
Q

where is prothrombin found?

A

bound to the external surface of platelets, linked to the negatively charged phospholipids of the membrane by Ca2+ ions

68
Q

what is the role of γ(gamma)-carboxyglutamates?

A

side-chains of these residues bind Ca2+ strongly, which can then act as the ‘glue’ to bind prothrombin to the platelet phospholipid.

69
Q

what are γ(gamma)-carboxyglutamates formed from?

A

Vitamin K-dependent enzyme reaction

70
Q

what is the role of TPA?

A

Tissue-type plasminogen activator
TPA adheres to clot and binds plasminogen- targets activity to correct place
Serine protease activity of TPA cleaves plasminogen to active plasmin
Plasmin digests fibrin clot to small peptides

71
Q

what does the presence of enzymes in the blood indicate?

A

tissue in which they are normally found are damaged by disease or injury

72
Q

where is enzyme activity measured?

A

in serum

73
Q

where is enzyme activity measured?

A

in serum

74
Q

what does the presence of amylase indicate?

A

acute pancreatitis

75
Q

what does the presence of alkaline phosphatase indicate?

A

liver disease/ rickets

76
Q

what does the presence of lactate dehydrogenase indicate?

A

MI

77
Q

what does the presence of cytosolic enzymes indicate?

A

slight damage

78
Q

what does the presence of mitochondrial enzymes indicate?

A

severe damage

79
Q

why is time after injury crucial in enzyme measurements?

A

different half-life in the blood stream

80
Q

what is the standard measurement for enzyme activity?

A

International units

81
Q

what is 1 IU equivalent to?

A

amount of activity that will convert 1 micromole (µmole, or 10^-6 moles) of substrate per minute under standard defined conditions

82
Q

what is tested in a standard LFT?

A 
Albumin
AST
transaminases (GOT/GPT)
alkaline phosphatase
bilirubin
prothrombin time
GGT
83
Q

what does albumin indicate?

A

general loss of secretory function, but decrease could also be due to kidney disease

84
Q

what does AST indicate?

A

Acute liver damage, but could originate from RBCs or muscle, so not liver-specific

85
Q

what do transaminases indicate?

A

If high but with normal ALP, liver necrosis. Elevated levels could indicate alcohol toxicity, viral infection, cancer, others. If >1000 IU/L could be paracetamol poisoning and/or severe/sudden liver failure

86
Q

what does ALP indicate?

A

If elevated, could indicate large bile duct obstruction. Caution: also high in Paget’s Disease of Bone, growing kids and 3rd trimester pregnancy

87
Q

what does bilirubin indicate?

A

Direct bilirubin is conjugated for excretion in the bile- if normal, jaundice results from problem upstream of the liver (some form of haemorrhage causing high total bilirubin). Elevated direct bilirubin indicates normal conjugation function in the liver, but failure to excrete (bile duct obstruction).

88
Q

what does prothrombin time indicate?

A

slower clotting time indicates failure of the liver to synthesise and secrete blood clotting proteins such as prothrombin. Also used to monitor warfarin usage and Vitamin K status

89
Q

what does GGT indicate?

A

Sensitive to minor changes in liver functions, more ‘liver specific’ than transaminases. Raised by chronic alcohol/drug abuse.

90
Q

what is acid phosphatase indicative of?

A

prostatic carcinoma

91
Q

what ard the three marker enzymes for MI?

A

CK: Creatine kinase
GOT: Glutamate-oxaloacetate transaminase
LDH: Lactate dehydrogenase

92
Q

what are isoenzymes?

A

catalyse the same reaction, but often show a preference for driving a reaction in one direction over another, and display different properties such as substrate affinity (reflected in different Km values). Isoenzymes may also be regulated in differently, and often show varying sensitivity to inhibitors.

93
Q

how many different isoforms of lactate dehydrogenase are there and what are they?

A 
5
H4 -------  Heart (LDH1)
H3M ----- Monocytes/macrophages (LDH2)
H2M2 ----- Lungs (LDH3)
HM3 ------ Kidney, pancreas (LDH4)
M4 ------- Liver, skeletal muscle (LDH5)
94
Q

what are the two different subunits found in LDH?

A

H’ abundant in heart and blood cells, ‘M’ in muscle, kidney, liver

95
Q

what does LDH catalyse?

A

the conversion of pyruvate to lactate (or vice versa), using NADH as a co-factor

96
Q

what is paracetamol overdose treated with?

A

N-acetylcysteine/methionine- precise dose required

IMS (13 decks)

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