CH 5 (LG)

Question 1

enzyme inhibition

Answer 1

• a kind of regulation
• dissociation constant (Kd) for this kind of interaction is the same as the inhibition constant (Ki)
• when an inhibitory molecule is on the enzyme, it can have multiple effects
• the manner in which inhibitors work varies

E + I EI
Kd = Ki = [E][I] / [EI]

Question 2

Inhibition

Answer 2

4 ways:

1) competitive inhibition (classical and non classical)
2) uncompetitive inhibition
3) noncompetitive inhibition
4) mixed inhibition

Question 3

Competitive Inhibition

Answer 3

a) classical competitive inhibition –>
Substrate (S) and the inhibitor (I) compete for the SAME site on the enzyme

b) nonclassical competitive inhibition –>
The binding of substrate (S) at the active site prevents the binding of inhibitor (I) at a separate site and vice versa
(S and I binds at DIFFERENT sites)

• -> the most commonly encountered inhibitors
• once inhibitor is bound to the enzyme, substrate cannot bind
• binding of substrate also prevents inhibitor from binding
• -> they COMPETE for binding of the same active site (most common)

Question 4

competitive inhibition graph

Answer 4

• formation of the EI complex inhibits substrate binding and therefore inhibits product formation
• -> this can be overcome by adding more substrate to increase [S]
• -> with sufficient [S], enzyme can be saturated, therefor Vmax (point in Y axis) remains the same
• What changes? Km (point in x-axis)

Question 5

inhibitors

Answer 5

• can be analogous (similar) to the substrate for the enzyme
• similar in structure but cannot be converted to product (do no react)
  Example:
• succinate dehydrogenase (E) converts succinate (S) —> to fumarate (P)
• malonate acts as the competitive inhibitor

Question 6

Uncompetitive Inhibition

Answer 6

• inhibitor (I) binds ONLY to the enzyme substrate (ES) complex, preventing the conversion of substrate (S) to product (P)
• uncompetitive inhibitors only bind to the ES complex
• -> they do not compete with substrate for binding to the enzyme
• -> they decrease Vmax and Km
• -> lower Vmax (upward direction of Y axis points), lower Km (left direction of x axis points)
  
  • Why? inhibitor causes a shift toward complexes

Question 7

Noncompetitive Inhibition

Answer 7

• the inhibitor (I) can bind to either E OR ES.
• the enzyme becomes inactive when I binds.
• substrate (S) can still bind to the EI complex but conversion to product is inhibited
• -> noncompetitive inhibitors bind either E or ES complexes
• -> no substrate analogs, do not bind same site as substrate
• -> lowers Vmax, Km is unaffected
• -> likely functions by altering shape of E, inhibiting S–> P reactions

graph:
V max is lowered (Y axis upward direction)
Km (x axis) stays the same

Question 8

Mixed Inhibition

Answer 8

• most enzymes do not conform to the noncompetitive model wherein Km is unaffected
• -> those that AFFECT BOTH Km and Vmax as mixed inhibition models

Question 9

Effects on Enzyme Kinetics

Answer 9

observable changes can be visualized and determined experimentally on a given enzyme’s kinetics

–> this will help determine what kind of inhibition is occurring

Question 10

effects of reversible inhibitors on kinetic constants

Answer 10

1) competitive: I binds to E only –> Vmax same, raises Km
2) uncompetitive: I binds to ES only –> lowers Vmax and Km, ratio of vmax/km unchanged

3) noncompetitive ( I binds to E or ES)
- -> lowerts Vmax, Km same

Question 11

daily use of competitive inhibitors

Answer 11

• products like Roundup use a competitive inhibitor as the main active ingredient
• in this case, it is glyphosate
• inhibits 5-enolpyruvylshikimate-3-phosphate synthase
• absorbed through leaves, not roots
• inhibits aa synthesis of Tyr, Trp, Phe in weeds and grasses

Question 12

Ibuprofen

Answer 12

• the OTC medicine is a competitive inhibitor of the enzyme cyclooxygenase (COX inhibitor)
• this enzyme is involved in many signaling events in mammalian cells including pain and inflammation

Question 13

Drug design

Answer 13

inhibitors can be of extremely valuable clinical use

• they also represent a lot of money for pharmaceuticals
• classically, it was a trial-and-error design

Question 14

rational drug design

Answer 14

• design specific drugs to fit specific enzymes

Question 15

Purine Neucleoside phosphorylase (E)

Answer 15

• degradative reaction betweem phosphate and nucleoside guanosine
• a rational drug design was implemented and the N9-bound group of guanosine was replaced
• chlorinated benzene ring binds the sugar binding site and acetate side chain binds phosphate binding site
• 100-times more potent inhibitor than any compound by trial and error

Question 16

Irreversible Enzyme Inhibition

Answer 16

• when a stable covalent bond is formed between the inhibitor and enzyme, it is termed as an irreversible enzyme inhibitor
• it works to take its target enzyme out of the game
• typically occurs via alkylation or acylation of side chains in the active site

example:
e-amino group of lysine
–> e-amino group of K reacts with an aldehyde group to form a Schiff base
–> reduction by sodium borohydride stabilizes the Schiff base via reduction

example: DFP + reactive serine
- -> nerve gas diisopropyl fluorophosphate (DFP) inactivates hydrolases with a reactive serine as part of the active site
- -> serine protease chymotrypsin is inhibited irreversibly by DFP
- -> the end result is an irreversibly inhibited chymotrypsin (E)

Question 17

Nerve gases

Answer 17

• originally developed for military use, nerve gases are toxic poisons
• major action is irreverisble inhibition of serine esterase acetylcholinesterase (AcHE)
• AchE hydrolyses the neurotransmitter Acetylcholine (Ach) returning muscle to resting state

Question 18

Regulation of Activity

Answer 18

• we want to regulate enzymes activity because and enzyme gone awry can cause damage to an organism

Question 19

ways to regulate enzymes include:

Answer 19

1) synthesis and degradation
2) reversible modulation
3) irreversible modulation

Question 20

regulated enzymes

Answer 20

• those that are regulated reversibly are called regulated enzymes (affecting rate)
• many are metabolic enzymes that respond to changes in the environment
• metabolic enzymes become MORE active when the [S] increases or [P] decreases
• conversely they become LESS active when [S] decreases or [P] increases
• -> regulating the first enzyme in a metabolic pathway saves energy

Question 21

Allosteric Regulation

Answer 21

• regulated enzymes can be controlled via noncovalent allosteric regulation or covalent modification
• in allosteric regulation, small molecules can affect the conformation of the enzyme and thereby activity

– is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme’s active site. The site to which the effector binds is termed the allosteric site.

• Hb + O2 was an example

Question 22

Cooperative binding

Answer 22

• allosteric enzymes typically work via cooperative binding
• the Sigmoidal Shape occurs because of transition from T-state to R-state
• without substrate, it is in the T state
• as substrate binds, it transitions to R state through conformational changes

Question 23

Allosteric Site

Answer 23

• the allosteric site is where the inhibitor or activator (allosteric modulator or effector) binds causing the conformation change
• the change is transmitted to the active site
• usually located on separate domains and even subunits

example:
allosteric enyme: phosphofructokinase-1
- (e.coli) involved on glycolysis
- catalyzes ATP-dependent phophorylation of Fructose 6-phosphate

• -> phosphoenolpyruvate: an intermediate near the end of the glycolytic pathway is formed
  
  • it acts as an allosteric inhibitor of E.coli Phosphofructokinase-1
  • if its concentration rises, it means the pathways is blocked and this inhibits beginning of the pathway

Question 24

phosphofructokinase-1

Answer 24

• acts in one of the first steps of glycolysis
• ADP acts as an allosteric activator
• glycolysis makes ATP (not enough ATP = we need ATP)
• there are four binding sites (homotetrameric enzyme)
• -> when an ADP binds the regulatory site, it assumes an R-conformation
• -> what does that means for affinity for substrate? higher
• -> when phosphoenolpyruvate binds the same site, it assumes a T-conformation
• -> what does that mean for affinity for substrate? lower

Question 25

allosteric enzyme

Answer 25

• activators can affect Vmax, Km, or both
• the activities of these enzymes are changed by metabolic inhibitors or activators
• allosteric regulators bind NONCOVALENTLY
• typically these enzymes are multisubunit proteins
• usually has at least one substrate for which V0 vs [S] is sigmoidal

Question 26

Alteration of the Sigmoidal Curve

Answer 26

just E: black sigmoidal curve (middle/ control)
E + activator: blue, above black E curve
E + inhibitor: red, below black E curve

Question 27

Altering conformations

Answer 27

• addition of an inhibitor can affect the transition of the enzyme between its T and R states
• raises the apparent Km and lower its activity
• increase the proportion of enzyme in T state

Question 28

Concrete Model

Answer 28

• developed to explain cooperative binding of identical ligands
• assumes one binding site on each subunit
• it assumes when ligand binds and one site changes, the others change too (from T to R)
• can include allosteric regulation by the same principle
• based on observed structural symmetry of regulatory enzymes
• suggests all subunits in the multiunit protein have the same conformation
• 2 conformations:
  1) active R (relaxed) conformation which binds substrate tightly
  2) inactive T (tight, taut) conformation, which binds substrate less tightly

the conformations of all subunits change simultaneously = both subunits change conformation from the inactive T conformation to the active R conformation at the same time or vice versa

Question 29

Sequential model

Answer 29

• general model that allows for subunits to exist in different states
• proposes and induced-fit model: changing conformation of neighboring subunits
• allows for high and low-affinity conformations

Question 30

Covalent modification

Answer 30

• these occur via COVALENT attachment or removal of groups on the peptide chain
• usually slower than allosteric regulation
• must be reversible
• believed to free in T or R via modification
• most common in phosphorylation (S, T, Y, H)