lecture 10: enzyme inhibition

bromelain

cysteine protease - has little preference for specific protein sequences and cleaves at many amide bonds

• in pineapple

• 
  

trypsin

cleaves amide bond at C-terminal of Lys or Arg

thrombin

cleaves only between Arg-Gly bonds in certain recognition sequences

lock and key model

enzyme has a specific shape that binds to specific substrates

induced fit

enzyme undergoes conformational changes to fit different substrates

do enzymes change reaction equilibrium

no, only reaction rate

why does transglutaminase form amide bonds but proteases hydrolyze them?

enzymes catalyze both forward and reverse reactions

→ transglutaminase don’t FORM amide bonds, they are creating an amide exchange, which is a different reaction with different thermodynamics

how are enzymes regulated?`

1. changing enzyme bioavailability through protein synthesis (ex. RNA splicing)

2. catalytic efficiency through enzyme modifications

how do you change enzyme bioavailability

RNA processing, protein targeting, protein degredation

how do you change catalytic efficiency through protein modifications

binding of regulatory molecules, covalent modification, proteolytic processing

six ways cells regulate enzyme activity

1. binding of regulatory molecules at regulatory sites, including allostery: turns on and off the enzyme

2. isozymes: there are differnet times of enzymes for different tissues or organelles

3. covalent modification: phosphorylation, acetylation, etc

4. proteolytic activation: zymogens

5. proteolytic degradation: proteases and the proteosome

6. control of enzyme biosynthesis via gene expression

distribution of DFA drug approvals

enzymes - 17%

kinases - 16%

GPCR - 12%

transporter - 4.0%

nuclear R - 3.7%

others - 47.3%

irreversible enzyme inhibition

inhibitory molecule forms a covalent bond with the catlytic group in the active site

→ kills the enzyme

reversible enzyme inhibition

nonconvalent bonding of a small biomolecules or proteins to the enzyme

• inhibition is decreased by diluting the enzyme reaction

what are the 3 types of reversible enzyme inhibition

competitive, uncompetitive, and mixed

example of irreversible enzyme inhibition

DFP on catalytic Ser195 in chymotrypsin

competitive inhibition

inhibitor replaces the substrate at the active side

uncompetitve inhibition

inhibitor binds to another site of the enzyme AFTER THE SUBSTRATE IS BOUND and blocks product formation

mixed inhibition

the inhibitor bind to just enzyme or ES complex, doesn’t bind to active site

kinetic parameters for competitive inhibition

• can be overcome by increasing [S]

• V_max is unchanged, but K_m increases

what is K_{m, app}?

K_m when there is an inhibitor; strength of the inhibitor

• equation only applies to competitive inhibitors

how to calculate V_o

V_o = K₂ [ES]

K_I

how to calculate [ES]

kinetic parameters for uncompetitive inhibition

K_m is constant and V_max decrease

→ but K_{m, app} decreases

mixed inhibition

lowers V_max and may increase or decrease K_m

• K_m can also stay the same → special case their the binding of the substrate doesn’t influence the inhibitor binding

noncompetitive inhibition

K_i = K_i’ → where K_m stays the same in mixed inhibition

equations for kinetic parameters of different types of inhibition

K_m

strength of the ES complex

what will change K_m

something that will change binding affinity between the enzyme and the substrate

ATCase

allosteric regulation

• has an active and inactive from switched on by ATP and switched off by CTP

• inhibited by the end product of its pathway

ATCase kinetics

sigmoidal curve (cooperativity): the bidning of substrate to one active site increases activity at another active site

*do not follow Michaelis-Menten kinetics

If K₂ is as large as possible, can uncompetitive inhibitors bind?

yes, but they have to bind faster than produce release