Allosteric enzymes & Inhibitors Flashcards
what is an inhibitor?
Any molecule that acts to reduce the rate of an enzymatic reaction
act in different ways
small chemicals or larger polymers
drugs tend to be inhibitors of enzymatic reactions
what is a competitive inhibitor?
mimic the substrate to bind to the active site
can greatly increase the Km of the enzyme
How does competitive inhibition work?
differs from the substrate so cannot act in the sam way the substrate does
no reaction - EI complex just sits there
what is the dissociation constant?
Ki
units are in molar
can find the strength of the bonds between enzyme and inhibtor
Ki
[E]x[I] / [EI]
assumed that inhibitor I binds reversibly to the enzyme and is in rapid equilibrium with it
competitive inhibitor reduces the concentration of the free enzyme available for substrate binding
what is the effect on the michaeles-menten reaction in the presence of a competitive inhibitor?
[S] approaches infinity
v approaches Vmax
infinitely high [S] can overcome effects of competitive inhibitor
presence of I makes Km appear larger, binding of I and S to E are mutually exclusive
Curve shifts to the R
curve will eventually reach Vmax, have to add more substrate to outcompete inhibitor
Michaelis menten equation modified by a
v = Vmax [S] / aKm + [S]
Main effect is the introduction of a which is a change in Km
Ki measured
v = Vmax / aKm =[S] —> 1/v —> 1/[S] = 1/Vmax
What is uncompetitive inhibition?
inhibitor that binds to the enzyme-substrate complex but not directly to the free enzyme
doesn’t affect the catalytic function of the enzyme but not its substrate binding
what is Kis?
[ES] [I] / [ESI]
A dissociation constant
has units of M
enzyme-substrate-inhibitor complex is catalytically active
Kis measured
v = Vmax [S] / Km + a[S] —> 1/v —> 1/ [s] + a / Vmax
Both Km and Vmax are decreased
ratio Vmax / Km remains unchanged
effects of uncompetitive inhibition on Vmax are not reversed by increasing the substrate concentration
what is mixed inhibition?
binds to enzyme sites that participate in both substrate binding and catalysis
mixed inhibition measuring?
v = Vmax[s] / aKm + a’[s] —> 1/v —> 1/[s] + a’/Vmax
Special case pf non-competitive inhibition
Ki = Kis and a = a’
shows decrease in Vmax (reciprocal value) but Km remains constant
What is irreversible inactivation?
inhibitor binds irreversibly to an enzyme inhibitor is an inactivator
reduce effective level of [e]t and Vmax at all values of [s], not changing Km
double reciprocal lots similar to non-competitive inhibition
competitive inhibitor
Km - increases
Vmax- none
uncompetitive inhibitor
Km - decreases
Vmax - decreases
mixed inhibitor
Km - increases
Vmax - decreases
non-competitive inhibitor
Km - none
Vmax - decreases
what is a multi-subunit enzyme
enzymes made from more than one protein chain (quaternary structure)
chains can be identical or different
sometimes binding of substrate to one subunit influences the binding to other subunits
results in cooperatively between subunits
control of enzyme availability
controlled by the cell and are subject to dramatic changes over time
bacteria - minutes
higher organisms - hours
control of enzyme activity
directly regulated through structural alterations
allosteric mechanisms can cause large changes in enzymatic activity
what is the average size of an enzyme
300 amino acids
what is an allosteric effector?
any molecule that can bind to an enzyme away from the active site
changing the enzymes activity for its substrate
increase or or decrease the rate of reaction
how does this happen?
small changes in the structure in one part of the protein can be communicated throughout structure
affect the fit of the active site to the substrate
what is allostery?
where a chemical binds to an enzyme away from the active site, and this influences the kinetic properties of the enzyme
allosteric control of aspartate transcarbamoylase (ATCase)
ATCase catalyses first step in biosynthesis of pyrimidines
carbamoyl phosphate donates carbamoyl group in pathways leading to arginine as well as pyrimidines
aspartate & carbamoyl phosphate together give a product only used in synthesis of pyrimidine nucleotides
allosteric behaviour of ATCase
both substrates bind cooperatively to the enzyme
ATCase inhibited by cytidine triphosphate (pyrimidine nucleotide)
ATCase activated by adenosine triphosphate (purine nucleotide)
curve of ATCase
V vs [s] curve is sigmoidal rather than hyperbolic
consistent with cooperative binding
CTP decreases the enzymes catalytic rate
ATP increases the enzymes catalytic rate
CTP and ATP compatible to competitive inhibitors (affect Km and not Vmax)
ATCase regulation
coordinates rates of synthesis of purine and pyrimidine nucleotides
[atp]>[ctp]- ATCase activated
[ctp]>[atp]- ATCase inhibited
Structure of ATCase
300kDa
c6r6- C= catalytic subunit r= regulatory subunit
arranges in two sets of trimers of the catalytic subunits (c3) in complex with three sets of regulatory dimers(r2)
active site of ATCase
crystal structure in the presence of PALA
substrate bound to subunit interface
each trimer contributes three active sites to the complete enzyme
most active site residues belong to one subunit
ATCase has 2 quaternary forms, what are they?
T (tense) state - no substrate
R (relaxed) state - substrate bound
substrate binding transition
trimers separate along molecular threefold axis by 12 angstroms
regulatory dimer rotate clockwise
what is the basis for the sigmoidal curve?
one with a high Km (low affinity) for substrate T state
one with low Km (high affinity) for substrate R state
[S] increases, equilibrium from T to R state
steep rise in activity
what is co-operativity?
binding of of substrate increases likelihood of other binding - positive cooperatively
opposite for negative cooperatively
