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Chem390 > Enzymes > Flashcards

Enzymes Flashcards

1
Q

What are enzymes?

A

natural proteins that act as body’s catalysts
their molecular masses range from several thousand to several million Da
These are soluble and found in the cytosol of cells

They transform substrates into products via enzyme substrate complexes.

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2
Q

What is the effect of enzymes on the rate of substrate transformation?

A

Transformations take place with rate enhancements several orders in magnitude as compared to uncatalysed

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3
Q

How do enzymes work?

A

Substrate binds to the active site of enzyme (which is only comprised of a dozen or so amino acids)

Remainder of enzyme maintains integrity of active site and channels the substrate to the AS.

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4
Q

What is a cofactor?

A

aka co enzyme. usually an organic molecule or metal ion

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5
Q

Enzymes lower the activation energy of a reaction, how is this done?

A

provides reaction surface or cavity for binding substrates
brings reactants together
positions reactants correctly for correct transition state configuration
weaks bonds
may participate in mechanism via covalent interactions

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6
Q

What is the activation energy?

A

the difference in energy between the tarting material and the transition state

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7
Q

What determines the rate of a reaction?

A

the size of the activation

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8
Q

What do enzymes stabilise in order to lower the Activation energy?

A

the transition state

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9
Q

What is the binding specificity of an enzyme?

A

can be broad e.g. cytochrome p450 family
Or very specific where only one enantiomer can form the ES complex (or both enantionmers form the complex but only one is converted into the product)

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10
Q

What is enantiomeric specificty due to?

A

chirality of enzymes

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11
Q

How can the enantiomeric specificty of enzymes be utilised?

A

Can be used to resolute a racemic mixture. Most drugs are racemic mixtures (cheaper) this can explain why more expensive drugs (which only contain the pure active enantiomer) can be more effective.

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12
Q

What is reaction specficity?

A

Enzymes can distinguish between chemically identical protons

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13
Q

What is the active site?

A

generally located in a groove or cleft and acts as surface or focus for the reaction by bringing the substrates together and holding them in the best position for a reaction to take place. Enzyme active sites stabilise the reaction transition state and consequently lowers the activation energy for the transformation hence increasing rate. This is reversible.

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14
Q

What are the main roles of amino acids present in the active site ?

A

binding- binds substrate to active site
catalytic - involved in mechanism of reaction

This active site of many enzymes is conserved across many species.

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15
Q

What occurs with the removal or mutation of one of the key amino acids?

A

generally results in loss of activity

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16
Q

What kinds of interactions bind substrates to active sites of enzymes?

A 
ionic 
hydrogen bonding
VDW
dipole dipole interactions
induced dipole interactions
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17
Q

Are ionic binding interactions important for the binding of substrate to active site?

A

Yes.

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18
Q

What are dipole dipole interactions?

A

Dipole moment of substrate aligns with local dipole moment on the surface of the active site

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19
Q

What are induced dipole interactions?

A

aromatic ring can interact with ionic group like a quaternary ammonium ion.
The positive charge of ammonium ion distorts the pi cloud of the aromatic ring to produce a dipole moment such that the face of the aromatic ring is electron rich and the edeges are electron deficient

e.g. pyruvic acid binding to active site of lactate dehydrogenase

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20
Q

Why would we want to inhibit an enzyme?

A

deficiency or excess of a metabolite resulting in a disease state can be normalised by inhibiting the activtity of the enzyme involved

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21
Q

What are the modes of enzyme inhibition?

A

competitive/reversible inhibitors
irreversible inhibitors (Enzyme inactivator)
non-competitive reversible (allosteric inhibitors)

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22
Q

What are competitive reversible inhibitors?

A

A molecule can be designed which is similar to the natural substrate but binds the active site more strongly than the natural substrate
This competes with natural substrate for binding, blocking the active site and prevents the natural substrate from binding.

By clogging up the enzyme, its function is lowered.

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23
Q

What does the competitive (reversible) inhibitor not do?

A

undergo chemical reaction or is changed in anyway.

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24
Q

What happens with increased levels of natural substrate (in competitive (reversible) inhibition?)

A

Enzyme will resume function

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25
Q

What are the 4 different types of competitive inhibition used in drug design?

A

simple competitive inhibition
alternative substrate inhibition
transition state analog inhibition
slow, tight binding inhibition

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26
Q

what is simple competitive inhibition?

A

design of inhibitors whose structure resembles that of natural substrate of the enzyme.
Most common type
Inhibitor does not react with enzyme
Inhibitor only blocks the AS of the enzyme and prevents accessibility to the natural substrate

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27
Q

Whatis alternative substrate inhibition?

A

The molecule not only binds to the enzyme but also acts as a substrate. The inhibitor reacts with the enzyme and gets converted into products.
Disadvantageous if products formed are toxic

28
Q

What is transition state analog inhibition?

A

An inhibitor whose structure resembles the TS of the reaction

29
Q

What is slow, tight binding inhibition?

A

The equilibrium is reached slowly
substantial inhibition occurs when the concentration of enzyme and inhibitor are comparable. These inhibitors could bind either non covalently or sometimes even covalently with the AS

These are therefore the bridge between reversible and irreversible inhibitors

30
Q

What is the marked drug example of competitive enzyme inhibition?

A

Antihypertensive drugs.
Angiotensin converting enzyme is a Zn containing exopeptidase. It is non specific.

After SAR studies, it was found that C terminal proline, that prevented the enzyme from hydrolysing peptide and rendering it inactive

31
Q

why are inhibitors of carboxypeptidase A also inhibitors of ACE?

A

carboxypeptidase A is a metalloprotein. where the metal atom Zn lies in the active site, like ACE

From this knowledge, we developed Captopril which contains a proline and SH group as a common antihypertensive drug

32
Q

What is the mechanism of Carboxypeptidase A?

A

It hydrolyses a carboxyl terminal amino acid withan aromatic or bulky aliphatic side chain from a peptide chain. This is inhibited by L- benzylsuccinnic acid.

the L benzylsuccinnic acid lacks the peptide bond => no hydrolysis

33
Q

What were the disadvantages of captopril?

A

side effects. these included rashes, loss of taste, mucosal legions and persistent cough. ACE is used for the cleavage of other biologically important peptides so ace inhibition may potentiate the actions of these other peptides

34
Q

What are irreversible inhibitor?

A

these stop an enzyme altogether as binding is irreversible.
Increased levels of natural substrate cannot dislodge the inhibitor.

the inhibitor is an electrophilic drug which fits the active sitesand locates its electrophilic region close to the nucleophilic residues to form a covalent bond to the enzyme

35
Q

What are the most effective irreversible inhibitors?

A

compound which react with the nucleophilic -OH and -SH groups of amino acids like serine and cysteine which occur frequently in the enzyme AS

36
Q

What are non-competitive, reversible (allosteric) inhibitors?

A

Inhibitor has no structural similarity to the natural substrate. Increasing the amount of substrate has no effect on level of inhibition hence it is non competitive.

Allosteric inhibitors bind to a separate region of the protein and do not compete with substrate for the active site.

37
Q

what occurs with allosteric binding?

A

binding process changes the shape of the protein and masks the active site from the natural substrate so the substrate can no longer react.
inhibition is reversible if the inhibitor uses non covalent bonds to bind to the allosteric binding site

38
Q

What kind of enzymes can we design allosteric drugs to inhibit?

A

enzymes under feedback control. Drugs can be designed based on the structure of the product which binds to the allosteric site e.g. 6-mercaptopurine.

this is used in the treatment of leukaemia. Inhibits 1st enzyme involved in purine synthesis, blocks the synthesis and hence blocks dna synthesis

39
Q

What are allosteric binding sites important in?

A

the control and regulation of enzymes.
Small molecules can ind at a site remove from the active site, and result in changes in tertiary or quaternary protein structure which renders the enzyme inactive or more ative

40
Q

How is allosteric inhibition related to feedback control?

A

the product of the last enzyme in the biochemical series can allosterically inhibit the first enzyme to stop the pathway. this feed back control ensures cell does not produce more product than required.

41
Q

Why does the product act on the allosteric site and not the active site?

A

because it has been transformed from the original substrate and is not recognised by the active site therefore it must bind elsewhere

42
Q

What do enzymes provide to catalyse reactions?

A

binding interactions
acid/base catalysis of reaction
nucleophilic groups
cofactors

43
Q

What are binding interactions?

A

As allosteric interaction cannot be explained by a rigid lock and key principle, small molecules (which may be inhibitors, cofactors, substrates) binding to enzyme will force conformation of both molecules as a moulding process.

This is Koshland’s Theory of induced fit: where the substrate induces the active site to take up an ideal shape to accomodate it.

44
Q

What is acid base catalysis of enzymes?

A

This is provided by the amino acid histidine,
It is a weak base and can equilibrate between its protonated base and free base form,

It acts as a proton bank - can either donate or accept protons in the reaction mechanisms.

Important active sites, often hydrophobic, have a low concentration of water and an even lower concentration of protons .

45
Q

What nucleophilic groups are important in the catalytic role of enzymes?

A

Serine and cysteine are commonly present in active sites as they contain nucleophilic residues like OH and SH groups which can participate in the reaction mechanism.

They react with substrate to form intermediates which provide an alternative low energy pathway for the reaction. These intermediates would not be formed in the un catalysed reaction.

46
Q

What do serine and cysteine provide an alternative to? (Nucleophilic groups in the catalytic role of enzymes)

A

Water, for the initial step in hydrolysis of peptides.

Serine and cysteine are usually close to a histidine residue which is also involved in the catalysis of the reaction

47
Q

What is an example of nucleophilic groups taking part in the catalytic role of enzymes?

A

The mechanism of hydrolysis of peptides by 3C protease

48
Q

How is the hydrolysis of peptide carried out?

A

Histidine starts of in the acid form, and donates a proton to the substrate.
Following nucleophilic attack, a resonance stabilised intermediate is formed,
Histidine then acts as a base to accept a proton from the substrate, forming the uncharged tetrahedral intermediate.
The substrate is cleaved, leaving the free peptide

49
Q

What are transition-state analogues?

A

A compound which mimics the transition state structure of the reaction. Inhibitors that approximate the transition state structure bind more strongly to active site than the substrate or product.

This works well for enzyme reactions involving two substrates a two substrates linked in transition state leads to more binding interactions

50
Q

How does the enzyme normally produce the transition state structure?

A

Enzyme alters bond lengths, bond angles and charge distribution

51
Q

What are the requirements for transition state analogues?

A

Compounds must be stable, and can only resemble the real transition state of the reaction.

E.g. 5-fluorouracil which is used to treat breast, liver and skin cancer.
This targets thymidylate synthetase

52
Q

How does 5-fluorouracil work?

A

It is not the transition state analogue itself, but it is converted in theory to the fluorinated analogue of 2’-deoxyuridylic acid which then combines with the enzyme and tetrahydofolate in situ to form the transition state analogue.

53
Q

How is the thymidylate synthetase blocked?

A

The required loss of F+ cannot happen.

The ultimate result is that DNA synthesis is halted and replication and cell division is blocked

54
Q

What is the rate limiting step of cholesterol biosynthesis?

A

The reduction of HMGCoA to mevalonate

55
Q

What is menvinolin?

A

Yielded from the fungi aspergillus terreus and monascus ruber. Isolated by screening natural and product extracts

This is highly active.

56
Q

How is Mevinolin administered?

A

As the lactone prodrug.

Activity arises as the secondary alcohol acts as a transition state mimic

57
Q

What is a medicinal use of enzyme inhibitors?

A

Enzyme inhibitors against microorganisms.

58
Q

What is the concept behind using penicillin as an antibiotic?

A

Bacterial cells and viral cells have some unique enzymes which are crucial for survival of the microorganism.
These inhibit the transpeptidase enzyme responsible for bacterial cell wall synthesis.

59
Q

What was the first penicillin natural product to be isolated?

A

Penicillin G

60
Q

How does penicillin inhibit cross linking of peptidoglycans in the bacterial cell wall?

A

Shape of penicillin closely resembles that of D-ala-D-ala causing transpeptidase to react with penicillin instead of the polypeptide chain,

Enzyme is covalently linked to the drug (irreversibly acylated)
The new cell wall cannot be produced and the dividing bacterium cannot survive

61
Q

What are problems with penicillin?

A

Ineffective when taken orally due to acid sensitivity
Sensitive to β-lactamases produced by bacteria
Has a narrow spectrum of antibacterial activity

62
Q

Which penicillins are acid resistant?

A

Penicillin V: has a phenoxymethyl bond. (Which looks like Benzene-ether-C=O)

63
Q

What are penicillinase resistant penicillins?

A

Methicillin (acid sensitive)

Oxacillin, cloxacillin, flucloxacillin (acid resistant)

64
Q

Which antibiotics are broad spectrum?

A

Ampicillin, amoxycillin, carbenicillin

65
Q

Which enzymes inhibitors are important in the battle against viral infections?

A

Mainly protease inhibitors

E.g. Acyclovir for herpes and zidovudine for HIV

66
Q

What are examples of enzyme inhibitors against the human body’s own enzymes?

A

Inhibition of specific enzymes can depress the metabolism of neurotransmitters e.g. Monoamine oxidase inhibitors prolong the life of catecholamines like adrenaline, can act as a stimulant to treat low blood pressure.

E.g. Anticholinesterases can be used to treat Alzheimer’s disease

E.g. COX inhibitors like indomethacin used to treat rheumatoid arthritis

E.g. Capase enzymes implicated in process leading to cell death. Hence capase inhibitors potential treatment for stroke patients

Chem390 (21 decks)

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