Allosteric enzymes & Inhibitors

Question 1

what is an inhibitor?

Answer 1

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

Question 2

what is a competitive inhibitor?

Answer 2

mimic the substrate to bind to the active site
can greatly increase the Km of the enzyme

Question 3

How does competitive inhibition work?

Answer 3

differs from the substrate so cannot act in the sam way the substrate does
no reaction - EI complex just sits there

Question 4

what is the dissociation constant?

Answer 4

Ki
units are in molar

can find the strength of the bonds between enzyme and inhibtor

Question 5

Ki

Answer 5

[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

Question 6

what is the effect on the michaeles-menten reaction in the presence of a competitive inhibitor?

Answer 6

[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

Question 7

Michaelis menten equation modified by a

Answer 7

v = Vmax [S] / aKm + [S]

Main effect is the introduction of a which is a change in Km

Question 8

Ki measured

Answer 8

v = Vmax / aKm =[S] —> 1/v —> 1/[S] = 1/Vmax

Question 9

What is uncompetitive inhibition?

Answer 9

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

Question 10

what is Kis?

Answer 10

[ES] [I] / [ESI]

A dissociation constant
has units of M

enzyme-substrate-inhibitor complex is catalytically active

Question 11

Kis measured

Answer 11

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

Question 12

what is mixed inhibition?

Answer 12

binds to enzyme sites that participate in both substrate binding and catalysis

Question 13

mixed inhibition measuring?

Answer 13

v = Vmax[s] / aKm + a’[s] —> 1/v —> 1/[s] + a’/Vmax

Question 14

Special case pf non-competitive inhibition

Answer 14

Ki = Kis and a = a’
shows decrease in Vmax (reciprocal value) but Km remains constant

Question 15

What is irreversible inactivation?

Answer 15

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

Question 16

competitive inhibitor

Answer 16

Km - increases
Vmax- none

Question 17

uncompetitive inhibitor

Answer 17

Km - decreases
Vmax - decreases

Question 18

mixed inhibitor

Answer 18

Km - increases
Vmax - decreases

Question 19

non-competitive inhibitor

Answer 19

Km - none
Vmax - decreases

Question 20

what is a multi-subunit enzyme

Answer 20

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

Question 21

control of enzyme availability

Answer 21

controlled by the cell and are subject to dramatic changes over time
bacteria - minutes
higher organisms - hours

Question 22

control of enzyme activity

Answer 22

directly regulated through structural alterations
allosteric mechanisms can cause large changes in enzymatic activity

Question 23

what is the average size of an enzyme

Answer 23

300 amino acids

Question 24

what is an allosteric effector?

Answer 24

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

Question 25

how does this happen?

Answer 25

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

Question 26

what is allostery?

Answer 26

where a chemical binds to an enzyme away from the active site, and this influences the kinetic properties of the enzyme

Question 27

allosteric control of aspartate transcarbamoylase (ATCase)

Answer 27

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

Question 28

allosteric behaviour of ATCase

Answer 28

both substrates bind cooperatively to the enzyme

ATCase inhibited by cytidine triphosphate (pyrimidine nucleotide)
ATCase activated by adenosine triphosphate (purine nucleotide)

Question 29

curve of ATCase

Answer 29

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)

Question 30

ATCase regulation

Answer 30

coordinates rates of synthesis of purine and pyrimidine nucleotides
[atp]>[ctp]- ATCase activated
[ctp]>[atp]- ATCase inhibited

Question 31

Structure of ATCase

Answer 31

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)

Question 32

active site of ATCase

Answer 32

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

Question 33

ATCase has 2 quaternary forms, what are they?

Answer 33

T (tense) state - no substrate
R (relaxed) state - substrate bound

Question 34

substrate binding transition

Answer 34

trimers separate along molecular threefold axis by 12 angstroms
regulatory dimer rotate clockwise

Question 35

what is the basis for the sigmoidal curve?

Answer 35

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

Question 36

what is co-operativity?

Answer 36

binding of of substrate increases likelihood of other binding - positive cooperatively
opposite for negative cooperatively