Enzymes 3 - inhibition & regulation Spencer Flashcards
T/F covalent interactions bind reversible inhibitors to enzymes
false - non-covalent interactions
how does reversible inhibition alter michelis-menten kinetics?
Vm and Km can be altered into Vm apparent and Km apparent
what is the dissociation constant for the EI complex in competitive inhibition
Ki = [E][I] / [EI]
how does competitive inhibition change the michelis-menten plot?
same Vm
increased Km by a factor of 1+[I]/Ki
so lower hyperbolic takes longer to reach same Vm
how does competitive inhibition change the lineweaver-burke plot?
same Vm so same y-intercept
higher Km so decreased x-intercept and increased slope (lower catalytic efficiency)
how does competitive inhibition change catalytic efficiency?
decreases it
higher Km so kcat/Km is lower
what is the michelis-menten equation for a competitively inhibited enzyme reaction?
v0 = VmS / [Km(1+I/Ki) + S]
where Ki = dissociation constant for EI
how does competitive inhibition work?
inhibitor mimics shape and structure of substrate or transition state but lacks functionality for reaction – competes with substrate at the binding site.
T/F competitive inhibitors include both substrate analogs and transition state analogs
true
which are more potent inhibitors, substrate analogs or transition state analogs?
transition state analogs - bind more tightly to enzyme
what kind of reversible inhibitors bind to free enzyme only
competitive
what kind or reversible inhibitors bind to the ES complex only
uncompetitive
what kind of reversible inhibitors bind to E or ES complex
noncompetitive
what kind of reversible inhibitor binds only after substrate is bound?
uncompetitive
what is the michelis-menten equation for uncompetitive inhibition?
v0 = Vm/(1+I/Ki)S / [Km/(1+I/Ki) + S]
how does uncompetitive inhibition alter the michelis-menten plot?
lower Vm
lower Km
slopes are similar at low [S] (no gap)
how does uncompetitive inhibition alter the lineweaver-burke plot?
slopes are same (Km/Vm is same)
1/Vm increases (y-int up)
-1/Km more negative (x-int left)
how does uncompetitive inhibition alter catalytic efficiency?
does not alter it
kcat and Km decrease by same factor
just like taking E out of system, not affecting catalytic mechanism
which kind of reversible inhibition is the rarest, competitive, uncompetitive, or noncompetitive?
uncompetitive
how do uncompetitive inhibitors work?
bind to ES complex and hinder reaction (apparently increase E + S affinity by taking ES out of solution and driving reaction right)
how does noncompetitive inhibition work?
bind to allosteric site and inhibit catalytic mechanism (does not affect E + S binding)
how does noncompetitive inhibition alter the michelis-menten plot?
Vm is decreased
Km is same
slopes diverge from start (gap)
what is the michelis-menten equation for non-competitive inhibition?
v0 = Vm/(1+I/Ki)S / (Km + S)
how does noncompetitive inhibition alter the lineweaver-burke plot?
x-intercept remains same (-1/Km is same) y-intercept up (1/Vm increases) slope increases (Km/Vm increases)
how does noncompetitive inhibition affect catalytic efficiency?
decreases it
kcat down
Km same
kcat/Km down
which type of reversible inhibition does not affect E + S binding?
noncompetitive
only affects catalytic mechanism
which type of reversible inhibitor can be overcome by increasing [S]?
competitive
what are the main differences between michelis-menten plots for uncompetitive and non-competitive inhibition?
uncompetitive = no early gap, lower Km noncompetitive = early gap, same Km
conceptually, why does uncompetitive inhibition lower Km?
because [I] effectively takes some
which reversible inhibitor binds only to:
E
ES
E or ES
competitive
uncompetitive
noncompetitive
conceptually, why do uncompetitive inhibitors lower Km?
remove ES from solution, drive reaction right, increasing apparent E + S affinity
conceptually, why do noncompetitive inhibitors not lower Km?
remove E and ES from solution at equal rates, so does not drive reaction either direction by Le Chatlier’s principle
how does irreversible inhibition work?
inhibitor covalently binds to free E, often at active site but sometimes not, effectively removing [E] from solution
3 categories of irreversible inhibitors
group-specific
affinity labels
suicide inhibitors
how does irreversible inhibition affect Vm and Km?
decrease Vm
no change Km
what is the main difference between group-specific, affinity label, and suicide irreversible inhibitors?
specificity to the active site
- group-specific can bind active or allosteric site
- affinity labels bind active site
- suicide inhibitors bind active site; tricks enzyme into reaction that permanently modifies
which irreversible inhibitors are substrate analogs?
affinity labels and suicide inhibitors
group-specific reagents do not always bind active site
T/F allosteric enzymes do not fit the michaelis-menten model
true - usually sigmoidal v0 vs [S] relationship
what is a homotropic effector
a substrate that allosterically affects enzyme activity (usually increases it)
what is a heterotropic effector
an allosteric effector that is not the same as the enzyme substrate
T/F allosteric enzymes are always oligomers
false - they are Usually oligomers (interaction on one subunit affects others) but some monomeric allosteric enzymes do exist
how do regulatory proteins work in the case of PKA
regulatory proteins block active site of PKA until cAMP binds regulatory subunit and unblocks
what is reversible covalent modification
enzymes covalently modified by adding charged groups (phosphate, sulfate, acetate) that cause conf change an activation or inactivation (e.g. phosphorylation)
what is proteolytic activation
activating an enzyme by irreversibly cleaving part of the molecule away
reversible inhibition, irreversibile inhibition, allosteric control, regulatory proteins, reversible covalent modification, and proteolytic activation are all forms of…
enzyme inhibition and/or control
T/F enzymes under allosteric control are usually rate-determining enzymes in metabolic pathways
true
does allosteric regulation involve covalent or non-covalent binding?
non-covalent
T/F phosphorylation with a kinase is the reverse of dephosphorylation with a phosphatase
false - the net reaction is
ATP + H2O -> ADP + Pi
kinase does not generate H2O
phosphatase does not generate ATP
T/F target proteins can cycle in either direction from unphosphorylated to phosphorylated using either phosphatase or kinase
false - phosphatase and kinase only work in one direction each
T/F phosphorylation and dephosphorylation are each reversible reactions
false - k3 / kcat only, very little to no k4
T/F the overall phosphorylation/dephosphorylation cycle results in hydrolysis of ATP
true - ATP is not regenerated by dephosphorylation – Pi is the product
T/F the overall phosphorylation cycle has a free energy change greater than zero
false - ∆G < 0
ATP + H20 -> ADP + Pi
reactants are higher energy than products
if given a question gives v initial and v final for two different substrate concentrations with and without inhibitor, how do you know what kind of inhibitor it is?
vf/vi inhibited = vf/vi normal, noncompetitive
vf/vi inhibited < vf/vi normal, competitive
vf/vi inhibited > vf/vi normal, uncompetitive
what is difference between noncompetitive inhibitor and negative allosteric effector?
noncompetitive I = binds E or ES and inhibits reaction… does not reduce E + S affinity
neg allosteric eff = reduces E + S affinity
what is the shortcut michaelis-menten equation for uncompetitive inhibition?
v0 = VmS / [Km + S (1+I/Ki)]
irreversible inhibition most closely resembles which kind of reversible inhibition?
noncompetitive
Km is same, Vm is decreased (taking E out)
