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Metabolism > Enzymes > Flashcards

Enzymes Flashcards

1
Q

what are enzymes

A

biological catalysts which speed up rate of reaction, by lowering activation energy, without altering the final equilibrium of reactants and products

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2
Q

how do enzymes affect the final equilibrium of reactants and products

A

they dont; they remain unchanged

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3
Q

how do enzymes speed up rate of reactions

A

by lowering the activation energy required for reaction

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4
Q

roles of enzymes

A
  • speed
  • selectivity
  • specificity
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5
Q

how enzymes affect speed

A
  • enhance rate of reaction 10^6 - 10^14 times compared to uncatalysed reaction
  • some reactions are so fast they are only limited by the diffusion rate of substrate to active site
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6
Q

how do enzymes affect specificity

A

enzymes can be very specific
e.g
- glucosidase will hydrolyse glucose from an oligosaccharide, but will not hydrolyse galactose from a oligosaccharide
- glucose and galactose differ at only one chiral centre on the opposite side of the ring from point of hydrolysis
= great specificity

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7
Q

how do enzymes affect selectivity

A

enzymes will often catalyse only a single reaction and carry out that reaction stereoselectively

e. g
- a particular glucosyl transferase will only add glucose on to the 2 postition of another glucose
- it will not add glucose to the 3, 4, or 6 positions

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8
Q

effect of enzymes on activation energy of a reaction

A

enzymes help overcome the activation energy barrier

even if a reaction is energetically favourable (i.e goes from high to low free energy), there is till Ea to overcome

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9
Q

how do enzymes lower activation energy

A
  • often by taking a different route to get to the same destination
  • this may not occur via a simple transition state
  • there can be multiple intermediates that might not be present inthe uncatalysed reaction
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10
Q

Rate limiting step

A
  • always the highest transition energy COMPARED to the relative intermediate
  • this means it is not always the highest transition energy
  • look for segments where intermediate is lower in energy that intial reactants
  • will be the slowest step of the reaction
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11
Q

what is a key property of enzymes

A

defined 3D structure which enables binding of substrate in presence of certain AA, when arranged in a certain way which facillitae bindng at the active site

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12
Q

what is the active site

A

can be on the surface or inside an enzyme

location of chemical binding

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13
Q

steps of enzyme action

A
  • enzyme is active
  • specific substrate with correct stereospecificity will bind
  • enzyme-substrate-complex forms
  • catalysis occurs
  • enzyme-product-complex forms
  • product is released
  • enzyme is recycled
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14
Q

why does the product leave the enzyme

A

the product typically has a lower affinity for the enzyme than the substrate.
favourable characteristic as allows recycling of enzyme and product to be produced

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15
Q

substrate specificity

A
  • often enzymes will catalyse one type of reaction
    e.g alcholol dehydrogenase willl oxidise primary alcohols to aldehydes
    this is an example of group specificty because the enzyme acts on all primary alcohols
  • very few enzymes only act on one substrate
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16
Q

stereospecificity

A
  • if a substrate occurs naturally in two steriosomer forms, the enzyme concerned with its metabolism in the cell will usually only act on one isomer
  • hence some forms of subsrates are considered active and inactive
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17
Q

what determines specificity

A

presence of the active site, into which only the substrate with the correct shape and charge will fit

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18
Q

what has enzyme specificty lead to

A

systematic classification scheme

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19
Q

systematic classification scheme of enzymes

A
  • 6 main classes according to type of reaction they catalyse
  • each class is split into subgroups according to their substrate or source
  • each enzyme is identified by its own 4-digit number
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20
Q

6 classes of enzymes

A
  • oxidoreductases
  • transferases
  • hydrolyses
  • lyases
  • isomerases
  • ligases
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21
Q

type of reaction and example enzyme for: oxidoreductases

A
  • add O2 or remove H2
  • Lactate dehydrogenase
    = pyruvate -> lactate (by action of NADH)
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22
Q

type of reaction and example enzyme for: transferases

A
  • transfer of functional groups
  • alanine amino transferase
    = gulatmate+pyruvate -> alpha-ketoglutarate + L-alanine
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23
Q

type of reaction and example enzyme for: hydrolases

A
  • hydrolytic reactions

- Trypsin

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24
Q

type of reaction and example enzyme for: lyases

A
  • add groups to -C=C bonds
  • ATP-citrate lyase
    = citrate -> oxaloacetate + acetate
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25
Q

type of reaction and example enzyme for: isomerases

A
  • isomerisation reactions
  • phosphoglucose isomerase
    = G6P -> F6P
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26
Q

type of reaction and example enzyme for: ligases

A
  • form C-C or C-N bonds with ATP cleavage

- DNA ligase

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27
Q

enzyme structure

A
  • proteins and hence composed of one or more polypeptide chains, folded into a 3D complex
  • stabilised by many weak bonds
  • weak bonds are easily broken which causes disorganisation of structure
    = denatured
  • the active site of enzyme contains functional groups that stabilise the transition state of reactions
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28
Q

weak bonds of enzymes

A

H-bonds
electrostatic salt links
hydrophobic interactions

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29
Q

what happens when weak bonds are broken

A
  • easily broken e.g heat, pH
  • causes disorganisation of sturcutre
  • enzyme no longer has catalystic activity
    = inactive / denatured
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30
Q

hows is the transistion state of enzyme catalysis reaction stabilised

A
  • functional groups within the active site
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31
Q

Lock and key model for enzyme catalysis

A
  • specific for particular substrate
  • shape and electrostatic complimentation
    (elecrostatic = charge)
    = correct general concept but has now been modified because theory is too rigid; proteins are dynamic
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32
Q

what theory developed from the lock and key model

A

induced fit theory

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33
Q

Induced fit theory

A
  • enzymes will undergo a conformational change upon substrate binding
  • conformational change is induced by weak interactions with the substrate
  • confromational change allows the specific functional group within enzyme to position itself for catalysis
    = new enzyme conformation has enhanced catalysis reaction
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34
Q

what do conformational changes of enzymes effect

A
  • residues within the active site
    and/or
  • repositioning of entire domains
    = enhanced catalytic properties
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35
Q

what enzymes are conformational changes common

A

kinases which catalyse transfer of phosphate group and allow phosphorylation of substances
- substrates typically have c-terminal and n-terminal
(IGF-1 receptor is a tyrosie kinase)

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36
Q

stages of catalysis of peptide bond hydrolysis by chymotrypsin

A
  1. polypeptide substrate binds noncovalently with side chains of hydropobic pocket
    = enzyme substrate complex
  2. H+ is trasferred from the Ser to His within enzyme. Substrate forms a tetrahedral transtion state with the enzyme
    = first transtion state
  3. H+ is transferred to the C-terminal fragment of substrate, which is released as free peptide, by cleavage of C-N bond.
    The N-terminal peptide is bound to enzyme through acyl linakge to serine
    = acyl-enzyme intermediate
  4. water molecule binds to enzyme in place of departed free peptide
    = acyl-enzyme-H20-complex
  5. water molecule transfers its H+ to His of enzyme, and its -OH fragment to the remaining N-terminal fragment which again allows substrate to form a tetrahedral transtion state with the enzyme
    = second transition state
  6. second peptide fragment (N-terminus) is releaved. Acyl bond is cleaved, H+ is transfered from His back to Ser, and enzyme returns to initial state
    = product released and enzyme regenerated
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37
Q

exampe of enzyme with low specificity

A

Papain

= cysteine protease

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38
Q

what does papain act on and how

A
  • low substrate specificity
  • proteolytic enzyme
  • cleaves any peptide bond
  • very little regard for side chain of peptide
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39
Q

what is papain used as

A
  • meat tenderizer

- also breaks down protein toxins from jellyfish, bees, wasps and stingrays

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40
Q

example of enzymes with high specificity

A
  • typsin and thrombin

both are serine proteases

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41
Q

what does trypsin do

A
  • high specificity
  • serine protease
  • cleaves on the carbonyl side of argenine and lysine residues EXCEPT when followed by proline
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42
Q

what does thrombin do

A
  • high specificty
  • serine protease
  • cleaves Arg-Gly bonds in particular sequences in fibrinogen specifically
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43
Q

enzymes and their kinetic constants

A

K1 ______ K2
E + S -> ES -> E + Products
K-1

  • there is always only one S, becuase at the beginning of the reaction other substrates are in excess and would ot have changed concentration so are not a factor
  • there is no K-2 becuase not all reactions are reversible. Michaelis menten only cares for the very early stages of the reaction when not enough product has formed for reversible reactions and so K-2 is not a factor
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44
Q

Km equation

A

= K-1 + K2
—————
K1

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45
Q

Vmax equation

A

= K2 [E] total

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46
Q

during steady state approximation what happens to [ES]

A

remains constant

- same amount forming as dissapears

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47
Q

what happens to Km when K2 is very small

A

Km ≈ K-1
——-
K1

(like acid dissociation equations)

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48
Q

what does bein able to calculate Km allow

A

a proxy for affinity of substrate for particular enzyme can be calculated, providing that K2 is v small

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49
Q

what does small K2 mean in terms of the reaction

A

there is a slow turnover once the ESC has formed

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50
Q

what is Vmax

A

the limiting velocity of the reaction at a given [enzyme]

  • it is the rate obtained at satuaring levels of substrate and is sometimes reffered to as maximum velocty
  • units are Ms-1, but often quoted per weight enzyme per unit time
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51
Q

what is Km

A

michaelis menten constant. It has specific meaning:
- when [S] = Km, then velocity is Vmax/2

Km is the [S] at half the limiting velocity
- in steady state conditions, Km is a measure of the lifetime of the ES complex and gives an indication of the [substrate] required for significant catalysis to occur

  • units are M
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52
Q

what happens when [S] = Km

A

veolcity is Vmax/2

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53
Q

lower Km =

A

higher affinity of a substrate for specific enzyme

54
Q

higher Km

A

lower affinity of a substrate for a specific enzyme

55
Q

V0

A

initial velocity

56
Q

what is saturation kinetics

A
  • determination of inital velocity and dependenc of rate of substrate concentration
57
Q

amount of product increases as a functin of

A

time

58
Q

what does rate depend on when [S] &laquo_space;Km

A

rate depends linearly on [s]

- [S] is the limiting factor

59
Q

what does rate depend on when [S]&raquo_space; Km

A

rate is independent of [S]

- [E] is the limiting factor

60
Q

what is rate (v) dependent on

A

rate is linearly dependent on [E] total at any given value of [S]

61
Q

alternatives to michealis menten plot

A
  • Lineweaver- Burk plot
  • Eadie-Hofstee plot
  • Hanes plot
62
Q

what is the Lineweaver-Burk plot

A
  • double recipricol
  • rate equation is fliped to give straight line equation
  • slope = Km/Vmax
  • intercept = 1/vmax and -1/Km
63
Q

rate equation

A

Vmax[S]
v = —————–
[S] + Km

64
Q

lineweaver burk equation

A

1 Km 1 1
– = —- x – + —-
v Vmax [S] Vmax

y = mx + c

65
Q

what is the slope of LWB

A

Km/Vmax

66
Q

what are the intercepts of LWB

A

1/vmax

-1/ Km

67
Q

pros and cons of LWB

A

pros: clear way of identifying different types of inhibiton

cons:
- relies on extrapolation and can be influences by poor data distribution
- as [S] gets bigger, 1/[S] becomes smaller and data points start to cluser and never become -ve
= lose power in using lots of values

68
Q

what does eadie hofstee plot

A

v/[S] against v

69
Q

what are the intercepts of eadie hofstee plot

A

Vmax

Vmax/Km

70
Q

what does Hanes plot

A

[S]/v against [S]

71
Q

what are the intercepts of Hanes plot

A

Km/Vmax

-Km

72
Q

benefits of Hanes and Eadie hoftsee compared to LWB

A

both better for allowing proper extrapolation and better distribution of plots

73
Q

catalytic constant

A

Kcat
aka turnover number
= first order rate constant for the decomposition of the ESC to products
- units are S-1

74
Q

why is Km not sufficient

A

Km tells us affinity but that is not eough, by using Kcat (catalytic constant) which tells us the turnover number, we can determine catalytic efficiency

75
Q

KA

A

KA ≡ Kcat/Km

76
Q

≡

A

equivalent to

77
Q

specificty constant

A

AKA catalytic efficiency, KA ≡ Kcat/Km

-useful for examining enzyme kinetics when [S] <

78
Q

High KA

A

more efficient enzyme

79
Q

what is the second order rate constant of reaction at low [S]

A

KA

80
Q

What is the first order rate constant for the decomposition of ESC to products

A

Kcat

81
Q

what is the maximum value of the specificty constant

A

maximum KA is the diffusion rate constant, when diffusion of the reactant to the AS is the slowest step in the mechanism

82
Q

what does comparision of Ka for two substrates tell us

A

gives a measure of selectivity of the enzyme for one substrate over the other

83
Q

what does ranking by Kcat not account for

A

Km

84
Q

different sources of enzyme regulation

A
  • environmental
  • inhibiton of enzyme
  • allosteric binding
85
Q

why are enzymes regulated in industry

A
  • to be more profitable

- to find cures

86
Q

examples of environmental enzyme regulation

A
  • location
  • time
  • temperature
  • pH
87
Q

examples of enzyme inhibtion for enzyme regulation

A
  • reversible
  • irreversible
  • competitive
  • non-competitive
  • uncompetitive
88
Q

examples of allosteric binding for enzyme regulation

A
  • postive and negative effectors at different sites
  • multiple sub units
  • cooperative kinetics
89
Q

effect of temperature on enzyme-catalysed reaction

A
  • increased temp = increased likelihood of successful E and S collisions becuase of higher energy
  • maximum efficiency will be reached at a certain temp
  • after this temp, denaturation occurs
  • bell shape
90
Q

what is the plot for enzyme reaction rate with increased temperature

A

mostly follows arrhenius equation (increasing diagonal line)
however, complicated by denaturaton
- denaturation can occur at low and or high temperatures
= bell shaped curve

91
Q

in what ways does pH affect enzyme-catalysed reactions

A
  • direct effects
  • substrate effects
  • effects on the enzyme
92
Q

what are the direct effects of pH on enzyme-catalysed reactions

A
  • [H+] or [OH-] appear in the rate equations
93
Q

what are the substrate effects of pH on enzyme-catalysed reactions

A
  • changes in ionisation state of the substrate leads to additional acid/base catalysis
  • changes leading to altered binging to the enzyme and hence a change in Km
94
Q

what are the effects to enzymes from changng pH in enzyme-catalysed reactions

A
  • unfolding of the enzyme leading to complete activation

- changes in ionisation state of AS residues leading to change in Km or Vmax

95
Q

what is pKa

A

pKa is the pH value at which a chemical species will accept or donate a proton

96
Q

why is the optimal pH range of fumerase relatively small?

A
  • AS of fumerase has at least two titrable groups
  • one must be protonated and the other must be deprotonated for full activity
  • the pKa values of the groups ca be determined by extrapolating from the linear regions of the bell curve, becuase the two pKa values are sufficiently different
    (one favours acidic conditins, the other basic)
97
Q

effect of pH on amino acid side chains

A
  • side chains of AA can exist as either: depending on pH
    • charged polar
    • uncharged polar
  • the pK that is quoted is for the AA free in solution, but this will shift depending on environment

+ve nearby will lower the pK for basic and acidic AAs
-ve nearby will increase the pK for basic and acidic AAs

  • a more hydrophobic environment will favour the uncharged state
98
Q

rate of enzyme reactions is typically modulated by

A

amino acids

99
Q

action of reversible inhibitors

A

bind to the enzyme but can dissociate again

  • when they bind, enzyme has little or no activity
  • when they dissociate, enzyme activity is restored
  • reversible inhibition can be competitive, non-competitive or uncompetitive
100
Q

action of irreversible inhibitors

A

bind to the enzyme which is then permanenty dissociated

  • ofthen binding is slow
  • enzyme decays over time

e.g
di-isopropyl phosphofluoridate binds to chymotrypsin and chemically modifies the AS residue

101
Q

action of competitive inhibition

A
  • acts directly on the enzyme AS
  • inhibitor molecules have chemical structure similar to substrate
  • inhibitor binds to enzyme and replaces the substrate in the AS to prevent substrate binding

e.g
oxidation of succinate to fumarate is reversibly inhibited by malonate
- malonate binds preventing oxidation of succinate

102
Q

how does competitive inhibiton affect enzyme kinetics

A
  • increases Km
  • doesn’t affect Vmax
  • the inhibitor prevents the binding of substrate or vice versa, depending which is present in larger concentration
103
Q

how to overcome competitive inhibition

A

increase [S]

becuase when [S]&raquo_space; Km, rate is independent of Km

104
Q

action of non-competitive inhibition

A

when there is no binding of inhibitor to the active site

105
Q

how does non-competitive inhibition affect enzyme kinetics

A
  • doesnt affect Km
  • reduces Vmax
  • inhibitor does not affect substrate binding
  • inhibition is independent of [S] because the rate always depends on Vmax
106
Q

will increasing [S] overcome non-competitive inhibition

A

no, inhibition is independent of [S] because the rate always depends on Vmax

107
Q

will increasing [S] overcome competitive inhibition

A

yes becuase when [S]&raquo_space; Km, rate is independent of Km

108
Q

what inhibition reduces Km but doesnt affect Vmax

A

competitive

109
Q

what inhibition doesnt affect Km but reduces Vmax

A

non-competitive

110
Q

what inhibition reduces both Vmax and Km

A

uncompetitive

111
Q

action of uncompetitive inhibition

A

inhibitor binds only to the preformed ES complex

112
Q

how does uncompetitive affet enzyme kinetics

A

reduces BOTH Km and Vmax

  • Km is reduced as ES depleted and E+S equilibrium is restored
  • specificity constant is unaltered (Ka = Kcat/Km)
113
Q

how do allosteric enzymes work

A

function through reversible, non-covalent binding of compounds called allosteric modulators or allosteric effectors - these are usually small metabolites or cofactors

114
Q

what are allosteric enzymes

A

usually multi-subunit enzymes where the AS and the regulatory site are on different subunits

115
Q

action of allosteric modulators

A

can be stimulatory or inhibitory

increase or decrease affinity to increase or decrease enzyme activity

116
Q

homotrophic regulation

A

modulator is the substrate itself

117
Q

heterotrophic regulation

A

modulators are different from the substrate

118
Q

example of how allostery is useful

A

glucose -> F6P -> F1,6P -> PEP -> Pyruvate

  • conversion of PEP to pyruvate needs pyruvate kinase, and simulatenously converts ADP to ATP
  • BUT pyruvate kinase is inhibited by ATP
    however, pyruvate kinase is activated homotrophically by PEP and heterotrophically by F1,6P

complex mechanism of interaction means that [Substrate] and [modulator] can be altered to obtain set of +ve or -ve feedback loops for fine tuning of metabolism

119
Q

how do allosteric modulators work

A

induce conformational change within Enzyme structure to make them more or less active
usually:
T-state = less active form
R-state = more active form

  • the enzyme regulatory site is specific for its modulator
120
Q

are allosteric enzymes the same as uncompetitive inhibitors

A

NO

uncompetitive inhibitors do not induce conformational change

121
Q

difference between allosteric and non-allosteric enzymes

A

allosteric enzymes are larger and more complicated

122
Q

typical graph shape of [S] against V0 for non-allosteric enzymes

A

michaelis menten - rectangular hyperbola

123
Q

typical graph shape of [S] against V0 for allosteric enzymes

A

sigmoidal

- indicates positive and negative cooperativity

124
Q

how does allostery give a sigmoidal response

A

a result of combining the MM for the two ‘enzymes’

  • the less active T-state with high Km
  • the more active R-state with low Km
125
Q

how do regulators effect the sigmoidal response of allostery

A

sigmoidal response is maintained but will be shifted

126
Q

structure of Aspartate transcarbamoylase

A
  • allosteric enzyme
  • complex
  • quatnerary structure, C6R6
  • 6 catalytic subunits which form 2 trimers
  • 6 regulatory subunits which form 3 dimers

exists in R-state and T-state

127
Q

t-state

A

tense-state = less active

128
Q

r-state

A

relaxed state = more active

129
Q

ratio of Aspartate transcarbamoylase isomers

A

exists in R-state and T-state

  • T predominates in absence of substrate = slow catalysis possible
  • R predominates as substrate binds = fast catalysis possible

in abscence of substrate and regualtors, ATCase exists in equilibrium between the 2 states, with T state favoured by a factor of 200

130
Q

how does ATCase work allosterically

A

CTP is the end product of the synthetic pathway
- CTP inhibits action of ATCase by stabilising T-state
- CTP binding site is more than 50A away from the nearest AS
= allosteric binding

Metabolism (11 decks)

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