allosteric enzymes and inhibitors Flashcards
an inhibitor is
any molecule that acts to reduce the rate of an enzymatic reaction
competitive inhibitors
competitive inhibitors resembles the substrate so that is specifically binds to the active sites but differs from the substrate so that is cannot react as the substrate does
many drugs are..
competitive inhibitors since active sites are easy to target as the substrate is known so related molecules can be designed. e.g. sildenafil (viagra) mimics the substrate phosphodiesterase
example of competitive inhibitor
succinate hydrogenase , an enzyme that converts succinate to fumigate is competiitley inhibited by malonate –> which structurally resembles succinate
competitive inhibitors reduces the..
concentration of the free enzymes available for substrate binding –> smaller reaction
Ki
dissociation constant (unitsM)
when inhibitors bind covalently
the inhibition is permanent
to overcome competitive inhibition..
more substrate must be added e.g. outnumber the inhibitor, more likely enzyme will be filled with substrate than inhibitor
total enzyme conc
hf
in competitive inhibition only..
the Km is effected and the not the Vmax
[E]t
conc of all enzymes in system
[E]t =
[E] + [ES] +[EI]
[EI]
enzyme and inhibitor
competitive inhibition equation related to michaelis menten
line weaver burk and competitive inhibition
the varying slopes indicate the effect of the inhibitor on Km
the more competitive inhibitor
the higher the Km
High Km means
Max is reached at a higher [s]
High Km means
Max is reached at a higher [s]
worked example calculating Ki e.g. when the inhibitor conc is 50mM, the Km of the enzyme for its substrate increases 3 fold. What is the value of Ki (mM0
enzyme-inhibitor complex is..
catalytically inactive
an inactivator is..
when an inhibitor binds irreversibly to an enzyme
inactivators reduce… but do not effect..
effective levels of [E]t and therefore Vmax and does not change Km
Reagents that chemically modify specific amino acids can act as..
inactivators
uncompetitive inhibition
binds to the enzyme-substrate complex, but not the free enzyme -effects catalytic property but not substrate binding -only significant in multi substrate enzymes
uncompetitive inhibition does not effect
substrate binding but does affect catalytic activity
the inhibitor in uncompetitive inhibition binds to
directly to the enzyme-substrate complex but not the free enzymes
Kis
-inhibitor-substrate
Km and Vmax in uncompetitive inhibition
In uncompetitive inhibition, both Km as well as maximum velocity (Vmax) is influenced- decreased
how will Km and Vmax be affected in uncompetitive inhibition
decreased ration of Vmax/Km will stay the same
in uncompetitive inhibition increasing substrate conc will.
NOT reverse the effects
mixed inhibition
a mixed inhibitor binds tot he enzyme sites that participate in both substrate binding and catalysis Another mode of inhibition which is similar to that of non competitive inhibition but with an active ESI complex is termed the mixed inhibition. Such inhibition is common in metabolic feedback pathways. Enzymes showing this form of inhibition are generally allosteric in nature
Non-competitive - allosteric
Non-Competitive inhibition is where; the inhibitor binds to the different site in the enzyme. So, in contrast to competitive inhibition, they can bind along with substrate to the enzyme and here both EI and ESI is inactive Fig 3.
effects of inhibitors of Km and Vmax: competitive
Km- increases VMAX-nothing
effects of inhibitors of Km and Vmax: uncompetitive
Km- decreases Vmax-Decreases
effects of inhibitors of Km and Vmax: mixed
Km- increases VMAX- decreases
effects of inhibitors of Km and Vmax: non-competitive
-non -decreases
multi- subunit enzymes- uncompetitive
enzymes made up from more than one protein chain (quaternary)- chains can eb identical or diff -sometimes the binding of substrate to one subunit, influences the binding to other subunits–> co-opervtivity between subunits
average size of an enzyme
300 amino acids
binding of the substrate and catalysis is carried out by around..
20-30 amino acids
regulation of enzyme activity: 2
1) control of enzyme availability 2. control of enzyme activity
control of enzyme availability
the amount of enzyme in a cell depends on both its rate of synthesis and its rate of degradation- controlled by the cell
control of enzyme acuity
directly regulated through structural alterations that influence substrate binding affinity –> allosteric mechanisms can cause large changes in enzymatic activity
allostery
refers to the situation where a chemical binds to an enzyme away from its active site and this influences the kinetic properties of the enzyme
allosteric effectors can ..
increase rate of reaction - allosteric activator decrease rare of reaction- allosteric inhibitor
allosteric activator
increase rate of reaction
allosteric inhibitor
decrease rare of reaction
proteins are dynamic and this means that
small changes in the structure of the protein can be communicated throughout the entire structure
ATCase
-300KDA, has R and C subunit R=regualtory C=catalytic arranged as two sets of dimers of the catalytic subunit (c3) in complex with the sets of regulatory dimers (r2). Each R2 joins two C3 trimers -Stabilised by zinc ion bout to four cysteine residues -catalyses the first step in the biosynthesis of pyrimidine
allosteric behaviour of ATCase
it has been demonstrated that both substrate bind cooperatively to the enzyme -ATCase is allosterically inhabited by cytitidine triphosphate (CTP), a pyrimidine nucleotide -ATCase is allosterically activated by adenosine triphosphate (ATP), a purine nucleotide both ATO and CTP affect Km and Vmax and are comparable to competitive inhibitors
ATCase structure
-300KDA, has R and C subunit R=regualtory C=catalytic arranged as two sets of dimers of the catalytic subunit (c3) in complex with the sets of regulatory dimers (r2). Each R2 joins two C3 trimers -Stabilised by zinc ion bout to four cysteine residues
what: regulates the synthesis of purine and pyrimidine nucleotides –> needed in equal amounts for nucleic acid biosynthesis
ATCase
allosteric graph
sigmoidal
ATCase graph
sigmoidal rather than hyperbolic -consisten with co-operative bonding due to binding of substrate ti int active site favours the conversion of the entire enzyme to the resting state, increasing the activity at the other active sites
CTP
decreases the enzymes catalytic rate
ATP
increases the catalytic rate
example of two allosteric effectors
CTP and ATP on ATCase
active site of ATCase
each trimer contributes three active sites. Binding of PALA causes major changes in the quaternary structure
T state- tense state
one of the 2 distinct tertiary structures of ATCase, which predominated in the absence of substrate -favoured by CTP binding- less active
R state- relaxed state
another that predominates when substrates are bound -favoured by substrate binding- more active qCTP and ATP bind to..
T–> R state
-catalytic trimers operate along the molecular three fold axis by 12A
CTP binding to the regulatory subunit stabilises the..
T state -T state and R state ar win equilibrium- the binding of CTP shifts the equilibrium towards the T-state, decreasing the net enzyme activity and reducing the rate of N-carbamoylasparate generation
both CTP and ATP
the same site on the outer edge of the regulatory subunit 50 A away from catalytic site
how far away is the regulatory unit away from catalytic site
50A
CTP increases the stability of the
T state
ATP increases the stability of the
R state
co-opertivity
usually the binding substrate to one subunit increases the likelihood of other subunits substrate binding –> positive co-opertivity
rare co-opertivity
negative co-opertivity–> binding of a substrate reduced the likelihood of another substate binding to the catalytic unit
theory of co-operativity
assumes that the enzyme can exist in two states: 1)active R (relaxed) state 2)inactive T (tense) state
sequential model
binding of substrate to one subunit moves that subunit to the relaxed active state. it also makes it more likely that neighbouring subunits will bind
concerted model
entire enzyme will switch from he T to R state none go -likelihood of this ids determined by the number of substrate molecules bound -after the switch binding of the further substrate is more likely
which enzymes do not show M-M kinetics
multi-subunit enzymes–> co-operative enzymes usually show a concerted switch to more active state
