Biochem - allosteric enzymes and inhibitors

What are inhibitors and how do they work?

• Any molecule that acts to reduce the rate of an enzymatic reaction

• Inhibitors act in a number of different ways to achieve this effect

• Can either be small chemicals or larger polymers (including other proteins)

What are the four types of inhibitor?

• Competitive inhibitor

• Uncompetitive inhibitor

• Mixed inhibitor

• Non-competitive inhibitor

How do competitive inhibitors work chemically?

• Active sites are easy to target - substrate is known and so related molecules can be designed

• Well designed inhibitor can greatly increase the apparent Km of the enzyme

• Competitive inhibitor usually resembles the substrate so that it specifically binds to the active site - differs from the substrate so it cannot react as the substrate does

• Assumed the inhibitor binds reversibly to the enzyme and is in rapid equilibrium with it

• Enzyme-inhibitor complex is catalytically inactive

• A competitive inhibitor reduces the concentration of free enzyme available for substrate binding

• Ki is the equilibrium constant for inhibition (high = bad inhibition, low = good inhibition)

How do competitive inhibitors change Michaelis-Menten?

• When [S] approaches infinity, V approaches Vmax for any value of a

• Infinitely high concentration of substrate can overcome the effects of the competitive inhibitor

• Presence of I makes the Km appear larger as binding of I and S to E are mutually exclusive

• a is a measurement of how much Km has changed (1 = no change, 2 = doubled, 4 = quadrupled)

• aKm (apparent Km) is the same as a x Km (real Km)

How do you measure Ki with a competitive inhibitor?

• Measure rate at varied substrate concentration, with or without inhibitor

• Varying slopes indicate the effect of the inhibitor on the apparent Km

• Vmax remains constant

How does a Lineweaver-Burk plot change with a competitive inhibitor?

• X axis - 1/[S]

• Y axis - 1/v

• Gradient - aKm/Vmax

• Y intercept - 1/Vmax

• X intercept - - 1/aKm

• Trendlines all converge on the Y-intercept

How does an uncompetitive inhibitor work?

• Inhibitor binds directly to the enzyme-substrate complex but not to the free enzyme

• Enzyme-substrate-inhibitor complex is catalytically inactive

• Effect of uncompetitive inhibition are not reversed by increasing the substrate concentration

How does an uncompetitive inhibitor change Michaelis-Menten?

• Kis is a dissociation constant for the uncompetitive inhibitor - higher value = worse inhibitor/lower value = better inhibitor

• Both appKm and appVamx are decreased but the ratio of appVmax:appKm remains unchanged

• Apparent Km = Km/a’

• Apparent Vmax = Vmax/a’

How do uncompetitive inhibitors change the Lineweaver-Burk plot?

• X-axis - 1/[S]

• Y-axis - 1/v

• Gradient - Km/Vmax

• Y intercept - a’/Vmax

• X intercept - a’/Km

How do mixed inhibitors work chemically?

• Mixed inhibitor can bind to either the free enzyme (E) or the enzyme-substrate complex (ES)

How do mixed inhibitors change Michaelis-Menten?

• Need to calculate both Ki and Kis for the inhibitor

• Apparent Km = aKm/a’

• Apparent Vmax = Vmax/a’

Ki = ([E][I])/[EI]

Kis = ([ES][I])/[ESI]

How do mixed inhibitors change the Lineweaver-Burk plot?

• X axis - 1/[S]

• Y axis - 1/v

• Y intercept - a’/Vmax

• X intercept - -a’/aKm

• Trendlines cross in the negative portion of x but not on either axis

How does a non-competitive inhibitor work?

Binds both the free enzyme and the enzyme-substrate complex equally

How does a non-competitive inhibitor change Michaelis-Menten?

• Changing inhibitor concentration will change appVmax

• Will not change Km

How does a non-competitive inhibitor change the Lineweaver-Burk plot graphically?

• X axis - 1/[S]

• Y axis - 1/v

• X intercept - - 1/Km

• Y intercept - a’/Vmax

• Trendlines converge on the x axis in the negative portion of x

What causes irreversible inactivation of enzymes?

• Inhibitor binding irreversibly to an enzyme the inhibitor is classified as an inactivator

• Truly reduce the effective level of [Et] at all values of [S] without changing Km

• Similar plot to those of non-competitive inhibition

• Reagents that covalently modify specific amino acid residues can act as inhibitors

• DIPF reacts with the catalytic serine of serine proteases to inactivate the enzyme

What is an allosteric effector?

Any molecule that can bind to an enzyme away from the active site and change the enzyme’s activity for its substrate

• Effectors can either increase the rate of the reaction (allosteric activator) or reduce the rate of reaction (allosteric inhibitor)

• Proteins have inherent flexibility - change in the structure in one part of the protein can be communicated through the entire structure and can affect the fit of the active site to the substrate and the transition state

How can multi-subunit allosteric enzymes affect the catalytic activity of enzymes?

• Many allosteric enzymes are made from more than one protein chain

• Also cooperativity in some cases where substrate binding to one subunit affects binding of substrate to other subunits

What does ATCase catalyse?

• Catalyses the first step in the biosynthesis of pyrimidines

• Aspartate and carbamoyl phosphate react together to give a product that is used in the synthesis of pyrimidine nucleotides

How is ATCase inhibited?

• ATCase is allosterically inhibited by CTP (pyrimidine nucleotide)

• ATCase is allosterically activated by ATP (purine nucleotide)

• Different types of nucleotides control the production of nucleotides oppositely

• Substrates bind cooperatively to the enzyme

What do different concentrations of ATP and CTP cause the enzyme to undergo?

• [ATP]>[CTP] - activated to synthesis pyrimidine nucleotides until the concentration is equal

• [CTP]>[ATP] - inhibited by CTP and permits the purine nucleotide biosynthesis to balance the ATP and CTP concentrations

How is ATCase organised by subunits?

• Composition of c6r6

• r - regulatory subunit

• c - catalytic subunit

• Arranged as two sets of trimers of catalytic subunits with three sets of regulatory dimers - each r2 dimer joins two c3 trimers

How does ATCase change structure when activated and relaxed?

• Has 2 conformational states in equilibrium

• Tense (T) and relaxed (R) states

• T state is less active than R state

• All subunits are in the same state - T state is stabilised by CTP binding, R state is stabilised by substrate and ATP binding

How does ATCase not follow Michaelis-Menten kinetics?

• Protein formation as a function of substrate concentration produces a sigmoidal curve instead of a hyperbolic curve

• Binding of substrate to one active site favours the conversion of the entire enzyme into the R state, increasing the activity at the other active sites

• Active sites show cooperativity

How can ATCase be changed in order to adhere to Michaelis-Menten?

• Can be considered to be a mixture of two Michaelis-Menten enzymes

• One will be high Km

• One will be low Km

• As the concentration of substrate is increased, the equilibrium shifts from the T-state to the R-state

• Causes a steep rise in activity with respect to substrate concentration

• Only Km is affected by allostery

What is the effect of allosteric regulators on ATCase?

• CTP decreases the enzyme’s catalytic rate (shift to the right)

• ATP increases the enzyme’s catalytic rate (shift to the left)