Enzymes Flashcards
what are enzymes
biological catalysts which speed up rate of reaction, by lowering activation energy, without altering the final equilibrium of reactants and products
how do enzymes affect the final equilibrium of reactants and products
they dont; they remain unchanged
how do enzymes speed up rate of reactions
by lowering the activation energy required for reaction
roles of enzymes
- speed
- selectivity
- specificity
how enzymes affect speed
- 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
how do enzymes affect specificity
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
how do enzymes affect selectivity
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
effect of enzymes on activation energy of a reaction
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
how do enzymes lower activation energy
- 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
Rate limiting step
- 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
what is a key property of enzymes
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
what is the active site
can be on the surface or inside an enzyme
location of chemical binding
steps of enzyme action
- 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
why does the product leave the enzyme
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
substrate specificity
- 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
stereospecificity
- 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
what determines specificity
presence of the active site, into which only the substrate with the correct shape and charge will fit
what has enzyme specificty lead to
systematic classification scheme
systematic classification scheme of enzymes
- 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
6 classes of enzymes
- oxidoreductases
- transferases
- hydrolyses
- lyases
- isomerases
- ligases
type of reaction and example enzyme for: oxidoreductases
- add O2 or remove H2
- Lactate dehydrogenase
= pyruvate -> lactate (by action of NADH)
type of reaction and example enzyme for: transferases
- transfer of functional groups
- alanine amino transferase
= gulatmate+pyruvate -> alpha-ketoglutarate + L-alanine
type of reaction and example enzyme for: hydrolases
- hydrolytic reactions
- Trypsin
type of reaction and example enzyme for: lyases
- add groups to -C=C bonds
- ATP-citrate lyase
= citrate -> oxaloacetate + acetate
type of reaction and example enzyme for: isomerases
- isomerisation reactions
- phosphoglucose isomerase
= G6P -> F6P
type of reaction and example enzyme for: ligases
- form C-C or C-N bonds with ATP cleavage
- DNA ligase
enzyme structure
- 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
weak bonds of enzymes
H-bonds
electrostatic salt links
hydrophobic interactions
what happens when weak bonds are broken
- easily broken e.g heat, pH
- causes disorganisation of sturcutre
- enzyme no longer has catalystic activity
= inactive / denatured
hows is the transistion state of enzyme catalysis reaction stabilised
- functional groups within the active site
Lock and key model for enzyme catalysis
- 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
what theory developed from the lock and key model
induced fit theory
Induced fit theory
- 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
what do conformational changes of enzymes effect
- residues within the active site
and/or - repositioning of entire domains
= enhanced catalytic properties
what enzymes are conformational changes common
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)
stages of catalysis of peptide bond hydrolysis by chymotrypsin
- polypeptide substrate binds noncovalently with side chains of hydropobic pocket
= enzyme substrate complex - H+ is trasferred from the Ser to His within enzyme. Substrate forms a tetrahedral transtion state with the enzyme
= first transtion state - 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 - water molecule binds to enzyme in place of departed free peptide
= acyl-enzyme-H20-complex - 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 - 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
exampe of enzyme with low specificity
Papain
= cysteine protease
what does papain act on and how
- low substrate specificity
- proteolytic enzyme
- cleaves any peptide bond
- very little regard for side chain of peptide
what is papain used as
- meat tenderizer
- also breaks down protein toxins from jellyfish, bees, wasps and stingrays
example of enzymes with high specificity
- typsin and thrombin
both are serine proteases
what does trypsin do
- high specificity
- serine protease
- cleaves on the carbonyl side of argenine and lysine residues EXCEPT when followed by proline
what does thrombin do
- high specificty
- serine protease
- cleaves Arg-Gly bonds in particular sequences in fibrinogen specifically
enzymes and their kinetic constants
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
Km equation
= K-1 + K2
—————
K1
Vmax equation
= K2 [E] total
during steady state approximation what happens to [ES]
remains constant
- same amount forming as dissapears
what happens to Km when K2 is very small
Km ≈ K-1
——-
K1
(like acid dissociation equations)
what does bein able to calculate Km allow
a proxy for affinity of substrate for particular enzyme can be calculated, providing that K2 is v small
what does small K2 mean in terms of the reaction
there is a slow turnover once the ESC has formed
what is Vmax
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
what is Km
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
what happens when [S] = Km
veolcity is Vmax/2
lower Km =
higher affinity of a substrate for specific enzyme
higher Km
lower affinity of a substrate for a specific enzyme
V0
initial velocity
what is saturation kinetics
- determination of inital velocity and dependenc of rate of substrate concentration
amount of product increases as a functin of
time
what does rate depend on when [S] «_space;Km
rate depends linearly on [s]
- [S] is the limiting factor
what does rate depend on when [S]»_space; Km
rate is independent of [S]
- [E] is the limiting factor
what is rate (v) dependent on
rate is linearly dependent on [E] total at any given value of [S]
alternatives to michealis menten plot
- Lineweaver- Burk plot
- Eadie-Hofstee plot
- Hanes plot
what is the Lineweaver-Burk plot
- double recipricol
- rate equation is fliped to give straight line equation
- slope = Km/Vmax
- intercept = 1/vmax and -1/Km
rate equation
Vmax[S]
v = —————–
[S] + Km
lineweaver burk equation
1 Km 1 1
– = —- x – + —-
v Vmax [S] Vmax
y = mx + c
what is the slope of LWB
Km/Vmax
what are the intercepts of LWB
1/vmax
-1/ Km
pros and cons of LWB
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
what does eadie hofstee plot
v/[S] against v
what are the intercepts of eadie hofstee plot
Vmax
Vmax/Km
what does Hanes plot
[S]/v against [S]
what are the intercepts of Hanes plot
Km/Vmax
-Km
benefits of Hanes and Eadie hoftsee compared to LWB
both better for allowing proper extrapolation and better distribution of plots
catalytic constant
Kcat
aka turnover number
= first order rate constant for the decomposition of the ESC to products
- units are S-1
why is Km not sufficient
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
KA
KA ≡ Kcat/Km
≡
equivalent to
specificty constant
AKA catalytic efficiency, KA ≡ Kcat/Km
-useful for examining enzyme kinetics when [S] <
High KA
more efficient enzyme
what is the second order rate constant of reaction at low [S]
KA
What is the first order rate constant for the decomposition of ESC to products
Kcat
what is the maximum value of the specificty constant
maximum KA is the diffusion rate constant, when diffusion of the reactant to the AS is the slowest step in the mechanism
what does comparision of Ka for two substrates tell us
gives a measure of selectivity of the enzyme for one substrate over the other
what does ranking by Kcat not account for
Km
different sources of enzyme regulation
- environmental
- inhibiton of enzyme
- allosteric binding
why are enzymes regulated in industry
- to be more profitable
- to find cures
examples of environmental enzyme regulation
- location
- time
- temperature
- pH
examples of enzyme inhibtion for enzyme regulation
- reversible
- irreversible
- competitive
- non-competitive
- uncompetitive
examples of allosteric binding for enzyme regulation
- postive and negative effectors at different sites
- multiple sub units
- cooperative kinetics
effect of temperature on enzyme-catalysed reaction
- 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
what is the plot for enzyme reaction rate with increased temperature
mostly follows arrhenius equation (increasing diagonal line)
however, complicated by denaturaton
- denaturation can occur at low and or high temperatures
= bell shaped curve
in what ways does pH affect enzyme-catalysed reactions
- direct effects
- substrate effects
- effects on the enzyme
what are the direct effects of pH on enzyme-catalysed reactions
- [H+] or [OH-] appear in the rate equations
what are the substrate effects of pH on enzyme-catalysed reactions
- 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
what are the effects to enzymes from changng pH in enzyme-catalysed reactions
- unfolding of the enzyme leading to complete activation
- changes in ionisation state of AS residues leading to change in Km or Vmax
what is pKa
pKa is the pH value at which a chemical species will accept or donate a proton
why is the optimal pH range of fumerase relatively small?
- 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)
effect of pH on amino acid side chains
- 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
rate of enzyme reactions is typically modulated by
amino acids
action of reversible inhibitors
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
action of irreversible inhibitors
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
action of competitive inhibition
- 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
how does competitive inhibiton affect enzyme kinetics
- increases Km
- doesn’t affect Vmax
- the inhibitor prevents the binding of substrate or vice versa, depending which is present in larger concentration
how to overcome competitive inhibition
increase [S]
becuase when [S]»_space; Km, rate is independent of Km
action of non-competitive inhibition
when there is no binding of inhibitor to the active site
how does non-competitive inhibition affect enzyme kinetics
- doesnt affect Km
- reduces Vmax
- inhibitor does not affect substrate binding
- inhibition is independent of [S] because the rate always depends on Vmax
will increasing [S] overcome non-competitive inhibition
no, inhibition is independent of [S] because the rate always depends on Vmax
will increasing [S] overcome competitive inhibition
yes becuase when [S]»_space; Km, rate is independent of Km
what inhibition reduces Km but doesnt affect Vmax
competitive
what inhibition doesnt affect Km but reduces Vmax
non-competitive
what inhibition reduces both Vmax and Km
uncompetitive
action of uncompetitive inhibition
inhibitor binds only to the preformed ES complex
how does uncompetitive affet enzyme kinetics
reduces BOTH Km and Vmax
- Km is reduced as ES depleted and E+S equilibrium is restored
- specificity constant is unaltered (Ka = Kcat/Km)
how do allosteric enzymes work
function through reversible, non-covalent binding of compounds called allosteric modulators or allosteric effectors - these are usually small metabolites or cofactors
what are allosteric enzymes
usually multi-subunit enzymes where the AS and the regulatory site are on different subunits
action of allosteric modulators
can be stimulatory or inhibitory
increase or decrease affinity to increase or decrease enzyme activity
homotrophic regulation
modulator is the substrate itself
heterotrophic regulation
modulators are different from the substrate
example of how allostery is useful
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
how do allosteric modulators work
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
are allosteric enzymes the same as uncompetitive inhibitors
NO
uncompetitive inhibitors do not induce conformational change
difference between allosteric and non-allosteric enzymes
allosteric enzymes are larger and more complicated
typical graph shape of [S] against V0 for non-allosteric enzymes
michaelis menten - rectangular hyperbola
typical graph shape of [S] against V0 for allosteric enzymes
sigmoidal
- indicates positive and negative cooperativity
how does allostery give a sigmoidal response
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
how do regulators effect the sigmoidal response of allostery
sigmoidal response is maintained but will be shifted
structure of Aspartate transcarbamoylase
- 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
t-state
tense-state = less active
r-state
relaxed state = more active
ratio of Aspartate transcarbamoylase isomers
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
how does ATCase work allosterically
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
