Lec 20 & 21 Enzyme Regulation

SL-3 Enzyme Regulation

Enzymatic reactions (thus products) are regulated to match ________

cell requirements

SL-3 Enzyme Regulation

achieved by regulating enzyme:

1) ______

2) _______

1) Abundance

2) Activity

SL-3 Enzyme Regulation

Abundance is controlled by ________

enzyme regulation is a natural ___________ phenomenon.

gene expression (not treated)

physiological

SL-4 Enzymatic reaction velocity

The velocity of a reaction is typically controlled by the concentrations of ______ and _______

substrates and cofactors

SL-4 Enzymatic reaction velocity

What are some cofactors?

metal ions or organic coenzymes that participate in some enzyme reactions

SL-5 Enzyme Regulation

Accumulation of product reduces ______ of reaction

velocity

SL-5

For conversion of substrate, S, to product, P, reaction velocity v is given by ________

d[P]/dt

SL-5

Keq = _____

Keq = [P]/[S]

SL-5

Once the ratio of [P]/[S] approaches Keq, no further reaction is apparent, because of the ___________

increased rate of the reverse reaction

SL- 5

Some enzymes may also be _______regulated by their product

allosterically

SL-6

What are the different ways to regulate an Enzyme?

1) Covalent modification

2) Zymogen (proenzyme) activation

3) Isozymes

4) Control by modulatory proteins

5) Allosterically

SL-7 Enzyme Regulation – covalent modification

Covalent modification of an amino acid side chain can _______ an enzyme (e.g. : Serine, Threonine, Tyrosine, Aspartate)

Covalent modification of an amino acid side chain can activate or inactivate an enzyme (e.g. : Serine, Threonine, Tyrosine, Aspartate)

SL-7

The enzymes that introduce and remove modifications can be ________

regulated (e.g. by allosteric control or by covalent modification)

SL-7

How can we control the generation of Products?

By controlling the ratios between inactive /active enzymes the generation of products can be controlled

SL-8 Enzyme Regulation – covalent phosphorylation

SL-8 Enzyme Regulation – covalent phosphorylation

What is the most prominent form of covalent modification in cellular regulation?

Reversible phosphorylation

SL-8 Enzyme Regulation – covalent phosphorylation

Phosphorylation is accomplished by _________

Phosphorylation is accomplished by protein kinases

SL-8 Enzyme Regulation – covalent phosphorylation

Each __________ targets specific proteins for phosphorylation

Each protein kinase targets specific proteins for phosphorylation

SL-8 Enzyme Regulation – covalent phosphorylation

Phosphoprotein phosphatases catalyze the _________

Phosphoprotein phosphatases catalyze the reverse reaction – removing phosphoryl groups

SL-8 Enzyme Regulation – covalent phosphorylation

________ and ____________ themselves are targets of regulation

Kinases and phosphatases themselves are targets of regulation

SL-9 Other Covalent Modification that Regulates Protein Function

• Several different chemical modifications of proteins have been discovered

• Only a few are used to achieve __________ regulation through __________ of an enzyme between active and inactive forms

• Several different chemical modifications of proteins have been discovered

• Only a few are used to achieve metabolic regulation through reversible conversion of an enzyme between active and inactive forms

SL-10

_________ is a prominent modification for the regulation of metabolic enzymes

Acetylation

SL-10

Acetylation of an _______ group on a _________ residue changes it from a ______ charged amino group to a _______

Acetylation of an ε-NH3 + group on a Lys residue changes it from a positively charged amino group to a neutral amide

SL-10

acetylation - This change may have consequences for __________ and thus _______

This change may have consequences for protein structure and thus function

SL-10

The acetylating enzyme is termed an _________ or ________

The acetylating enzyme is termed an acetyl-CoA-dependent lysine acetyltransferase or KAT (More than 30 KATs are known in mammals)

SL-10

___________ by KDACs (lysine deacetylases) reverse the effects

Deacetylation by KDACs (lysine deacetylases) reverse the effects

SL-10

Acetylation of metabolic enzymes is an important mechanism for ____________

Acetylation of metabolic enzymes is an important mechanism for regulating the flow of metabolic substrates (e.g: carbohydrates and fats)

SL-11

Synthesis of enzyme as an inactive precursor Activation requires specific __________

proteolytic cleavage

SL-11

The hormone insulin (responsible for glucose uptake) is made as an inactive _________ precursor (proinsulin).

Proteolytic removal of __________ generates the active form, (two chains and three disulfide bonds)

The hormone insulin (responsible for glucose uptake) is made as an inactive 86 amino acid precursor (proinsulin).

Proteolytic removal of residues 31-65 generates the active form, (two chains and three disulfide bonds)

SL-11

Proenzyme activation by protease cleavage is _________ (reversible / irreversible)

Proenzyme activation by protease cleavage is irreversible

SL-12 Proenzymes of the digestive tract

SL-13 Proenzymes of the digestive tract

SL-14 Enzyme regulation - Isozymes

____________ equivalent subunits (small primary sequence differences not identical) but _________ distinct subunits

Structurally equivalent subunits (small primary sequence differences not identical) but catalytically distinct subunits

SL-14 Enzyme regulation - Isozymes

SL-15 Enzyme regulation – Binding of regulatory proteins

The catalytic (C) subunit of cAMP-dependent protein kinase is kept in an _______ form by the _______ subunit. Binding of c-AMP to the __ subunit releases the active enzyme.

The catalytic (C) subunit of cAMP-dependent protein kinase is kept in an inactive form by the regulatory (R) subunit. Binding of c-AMP to the R subunit releases the active enzyme.

SL-16 Allosteric regulation

What is Allosteric regulation?

Activation or inhibition of enzyme activity by small molecules (metabolites) that interact non-covalently with the enzyme

SL-16

The ________ binds to a site other than the substrate binding site (allo = other)

The allosteric effector binds to a site other than the substrate binding site (allo = other)

SL-16

Reversible binding of an effector to the enzyme allows for __________ and thus _______ of enzymatic activity

Reversible binding of an effector to the enzyme allows for very rapid response times and thus rapid control of enzymatic activity

SL-17 Allosteric regulation

Example: the product (F) as an allosteric inhibitor of the first enzyme of the pathway (E1).

This is called ________

E1 is called _________

Allosteric ________ is also common

feedback inhibition

E1 is called regulatory enzyme: Where activity of the metabolic pathway is regulated

Allosteric activation is also common

SL-18

Regulatory enzymes are often subject to _______ regulation

allosteric

SL-18 Allosteric regulation – regulatory enzymes

Kinetic does not follow the _________ equation

Michaelis-Menten

SL-18 Allosteric regulation – regulatory enzymes

Properties of regulatory enzymes:

1. ________ kinetics

2. Often show _______

3. _______ substrate binding is a special case of allostery

4. Regulatory enzymes are _______

1. Sigmoid/S-shaped kinetics

2. Often show Cooperativity: Binding of S make binding of other S easier to same molecules

3. Cooperative substrate binding is a special case of allostery

4. Regulatory enzymes are oligomeric (it follows from cooperative kinetics, more than one substrate binding site)

SL-19 Allosteric regulation – regulatory enzymes

Properties:

• Inhibition by _________ inhibitor does not conform to normal inhibition patterns

• Allosteric effector (inhibitor or activator) usually has _____ structural similarity to the substrate

• Effector binds at a site ______ from the substrate binding site

• Regulatory effects alters distribution of ______ distribution and __________ changes occurring in the enzyme as a result of _________

• Inhibition by feedback allosteric inhibitor does not conform to normal inhibition patterns

• Allosteric effector (inhibitor or activator) usually has little/no structural similarity to the substrate

• Effector binds at a site remote from the substrate binding site

• Regulatory effects alters distribution of enzyme distribution and conformational changes occurring in the enzyme as a result of effector binding

SL-20 Symmetry model for allosteric regulation: the MonodWyman-Changeux (MWC) model

what are the 2 states of allosteric proteins?

Allosteric proteins can exist in two states: R (relaxed) and T (taut)

SL-20

In the MWC Model , all the subunits of an oligomer must be in the _________ state

In the MWC Model , all the subunits of an oligomer must be in the same state

SL-20 Symmetry model for allosteric regulation: the MonodWyman-Changeux (MWC) model

T state predominates in the absence of __________

S binds much tighter to ___ than to ____

In the absence of ligand, the two states are called _____ and ____

The equilibrium constant (L) for the T0/R0 equilibrium is ______ , that is T0 predominates

T state predominates in the absence of substrate S

S binds much tighter to R than to T

In the absence of ligand, the two states are called R0 and T0

The equilibrium constant (L) for the T0/R0 equilibrium is large, that is T0 predominates

SL-20 Symmetry model for allosteric regulation: the MonodWyman-Changeux (MWC) model

• The substrate affinity of each state of the enzyme is described by a ____________ constant: _________(for the relaxed form) and __________ (for the tense form)

• KT is much ______(smaller/greater) than KR. That is, R0 has a higher affinity for the substrate than T0

• The substrate binds with higher affinity to the less abundant form of the enzyme

• The substrate affinity of each state of the enzyme is described by a dissociation constant: KR (for the relaxed form) and KT (for the tense form)

• KT is much greater than KR. That is, R0 has a higher affinity for the substrate than T0

• The substrate binds with higher affinity to the less abundant form of the enzyme

SL-22 MWC Model - cooperativity

• Substrate binds preferentially to R0 . The substrate bound form is R1 •

• Binding lowers the abundance of R0

• Equilibrium between T0 to R0 conformationsis driven to R0

• R has >1 substrate binding sites

• Substrate binding increases the concentration of R (i.e. R1 + R0) •

• Therefore, the amount (number) of substrate binding sites increases : positive cooperativity

SL-23 MWC Model cooperativity

SL-24 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

Activator : positive effector

• _______ and _______ bind preferentially to the __ form

• _______ equilibrium is shifted towards ___

• Number of S binding sites _______(increases/decreases)

• L is _______ (increased/decreased)

• _______ the affinity of S binding and reduce _______

• Substrate (S) and activator (A) bind preferentially to the R form

• T0/R0 equilibrium is shifted towards R0

• Number of S binding sites increases (without any addition of S)

• L is decreased

• Increase the affinity of S binding and reduce cooperativity

SL-25 Quiz

In the presence of a positive effector the YS/[S] curve will:

A - Shift to the right

B - Shift to the left

C - Shift vertically

D – None of the above

B

SL-26 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

Activator : positive effector

• Substrate (S) and activator (A) bind preferentially to the R form

• T0/R0 equilibrium is shifted towards R0

• Number of S binding sites increases (without any addition of S)

• L is decreased

• Increase the affinity of S binding and reduce cooperativity

SL-27 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

Inhibitor : negative effector

• _________ binds preferentially to the ___ form

• T0/R0 equilibrium is shifted towards _____

• Number of S binding sites _______

• L is _______

• _______ the affinity of S binding and ________ cooperativity

• Inhibitor (I) binds preferentially to the T form

• T0/R0 equilibrium is shifted towards T0

• Number of S binding sites decreases (L is increased)

• Decrease the affinity of S binding and increase cooperativity

SL-28 The Monod-Wyman-Changeux (MWC) model: allosteric regulation – K systems and V systems

‘K systems’:

the concentration of substrate that gives half-maximal substrate binding (K0.5) changes in the presence of A and I.

Vmax does not change

SL-28 The Monod-Wyman-Changeux (MWC) model: allosteric regulation – K systems and V systems

‘V systems’

• In ‘V systems’, allosteric effectors change the Vmax and K0.5 remains the same

• R and T forms of the enzyme have the same affinities for S, but differ in their affinities for A and I and in their catalytic properties

• This regulation is important when the cellular concentration of S is much greater that K0.5

SL-29 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

• In the case of positive cooperativity,

  • the substrate acting as a ______ effector, in this case called a ________

• In the case of positive cooperativity,

  • the substrate acting as a positive effector, in this case called a positive homotropic effector

SL-29 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

A ligand other than the substrate that activates the binding of substrate is called a ________ effector or allosteric _______

A ligand other than the substrate that activates the binding of substrate is called a positive heterotropic effector or allosteric activator

SL-29 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

A ligand that Inhibit the binding of substrate is called a ________ effector or allosteric _______

A ligand that Inhibit the binding of substrate is called a negative heterotropic effector or allosteric inhibitor

SL-29 The Monod-Wyman-Changeux (MWC) model : allosteric regulation

The MWC model cannot explain _______ in substrate binding

The MWC model cannot explain negative cooperativity in substrate binding

SL-30 The Koshland-Nemethy-Filmer (KNF) model : allosteric regulation

• There is no equilibrium between different _________ of the enzyme with no ____

• S binding ______ a ________ change, and the subunits can adopt different ______

• S binding causes the other subunit to undergo a ____ change, to a form that has a higher or lower ____ for S. The model can explain________ cooperativity

• _______ activator works in the same way as S, but by binding to a different site

• _________ works by preventing the __________ change induced by ____ binding

• There is no equilibrium between different conformers of the enzyme with no S

• S binding causes a conformational change, and the subunits can adopt different conformations (unlike in MWC)

• S binding causes the other subunit to undergo a conformational change, to a form that has a higher or lower affinity for S. The model can explain both negative and positive cooperativity

• Allosteric activator works in the same way as S, but by binding to a different site

• Inhibitor works by preventing the conformational change induced by S binding

SL-31 Key differences between the MWC and KNF models cooperativity

MWC

• In MWC, there is a pre-existing equilibrium between ___ and ___ forms, in the absence of _____.

• Substrate or activator binds preferentially to the ___ form

• All subunits must be in the ______ conformation, there are ____(number) possible conformations

• All subunits change conformation _____, therefore mechanism is called the ______ or _______ model

• In MWC, there is a pre-exisiting equilibrium between R and T forms, in the absence of ligand.

• Substrate or activator binds preferentially to the R form

• All subunits must be in the same conformation, there are only 2 possible conformations All subunits change conformation together, therefore mechanism is called the concerted or symmetry model

SL-31 Key differences between the MWC and KNF models cooperativity

KNF

• In KNF, conformational change is induced by _________

• Conformational change can be transmitted to a _________ subunit

• __________ conformations are possible

• Subunits can be in _________ (same/different) conformations and change ___________, so mechanism is called sequential model

• In KNF, conformational change is induced by ligand binding

• Conformational change can be transmitted to a neighboring subunit

• Intermediate conformations are possible

• Subunits can be in different conformations and change sequentially, so mechanism is called sequential model