Final Exam Study Notes: Enzyme Inhibition and Regulation

Enzyme Inhibition

• Irreversible Inhibitors:

  • Bind very tightly to enzymes, sometimes covalently.

  • Powerful tools for elucidating enzyme mechanisms.

  • Resemble pure noncompetitive inhibition by decreasing the concentration of functional enzyme at all substrate concentrations.

  • V{max}$decreases,$KM remains unchanged.

  • Lineweaver-Burk (LB) plots intersect on the x-axis at -1/KM$. Modification of a side chain allows identification of the residue and$V{max} for uninhibited enzyme (E).

  • E + I
    ightharpoon Equilibrium E I
    ightharpoon k_{in} E I^*

  • Example: DIPF (Diisopropylphosphofluoridate)

    • Covalently modifies a serine residue essential for enzyme function, thus irreversibly inhibiting the enzyme.

    • Example: Acetylcholinesterase inhibition by DIPF.

      • Acetylcholinesterase breaks down the neurotransmitter acetylcholine (Ach) into acetic acid and choline.

Enzyme Regulation

• Controlling Enzyme Activity

  • Noncovalent modification (allosteric):

    • Binding occurs at a site other than the active site, affecting enzyme activity.

    • Active and regulatory sites are separate.

    • Involves small metabolites or cofactors.

    • Uses allosteric effectors like BPG.

  • Covalent modification:

    • Example: Phosphorylation vs. dephosphorylation.

    • Terminal phosphate group from ATP attached to the hydroxyl groups of Ser, Thr, or Tyr.

    • Done by enzyme called kinase.

    • Phosphate removed by enzyme called phosphatase.

  • Proteolytic activation:

    • Irreversible hydrolysis of peptide bonds.

    • Example: Digestive enzymes or protease activation of viral proteins.

• Controlling Enzyme Availability

  • Rate of production vs. degradation of enzyme ($E$) by the cell.

  • Influenced by the level of transcription.

Noncovalent Modification: Allosteric Regulators

• Allosteric effectors or modulators are generally small chemicals.

• Allosteric effectors can be positive, improving enzymatic catalysis, or negative, reducing enzymatic catalysis.

• Involves subunit-subunit interactions.

• Regulatory site is separate from the catalytic site.

• Allosteric regulators can be activators or inhibitors affecting enzyme conformation.

Kinetics of Allosteric Regulators

• Differ from Michaelis-Menten kinetics.

• Two conformations:

  • Tense (T) state: A compact, inactive form.

  • Relaxed (R) state: An expanded or open active form.

• Show sigmoidal kinetics, representing cooperative binding among multiple subunits.

• Allosteric regulators affect binding affinity (KM$) and turnover rate ($k{cat}).

Aspartate Transcarbamoylase (ATCase)

• Catalyzes the first step of synthesizing pyrimidines (CTP, etc.).

• Regulation is crucial to produce the precise amount of CTP needed by the cell.

• ATCase displays sigmoidal kinetics.

• Feedback Inhibition:

  • CTP (final product) is an allosteric inhibitor.

  • ATP is an allosteric activator.

  • The regulator binds to a site distinct from the active site.

Structure of ATCase (c6r6, 300kD)

• 12 polypeptide chains.

• 6 catalytic subunits (two trimers, $c_3$).

• 6 regulatory subunits (three dimers, $r_2$).

• Isolated catalytic trimer exhibits catalytic activity but no cooperativity.

• Isolated regulatory dimer binds ATP/CTP but has no catalytic activity.

ATCase: T-State (low activity) vs. R-State (high activity)

• The two states are in equilibrium, with the T state being favored.

• The T state has a low affinity for substrate and low catalytic activity.

• The R state is the most active form.

• All active sites are in the same state, either T or R.

• Binding of substrate disrupts the equilibrium in favor of the R state, a property called cooperativity.

• Allosteric interactions in ATCase are mediated by large changes in quaternary structure.

ATCase: Conformational Changes

• Binding of substrate to one catalytic subunit:

  1. Increases the substrate-binding affinity.

  2. Increases the catalytic activity of the other five catalytic subunits.

• Enzyme's cooperative substrate binding.

Allosteric Regulators Modulate the T-to-R Equilibrium

• CTP stabilizes the T state.

• Binding of CTP to the regulatory site of ATCase alters the T-to-R equilibrium in favor of the T state, decreasing net enzyme activity.

• ATP stabilizes the R state.

• ATP is a positive regulator of ATCase, altering the T-to-R equilibrium in favor of the R state, increasing net enzyme activity.

Unreactive Bisubstrate Analog Binds to R State

• Unreactive bisubstrate analog stabilizes the R state.

• Analog of reaction intermediate and potent competitive inhibitor.

• Regulatory dimers rotate/separate.

• Catalytic trimers open ~ 11°Å wide.

• Secondary structures are largely preserved during the transitions.

• Quaternary shift similar to Hemoglobin.

The T State and the R State

• The T state:

  • Has a low affinity for substrate.

  • Has low catalytic activity.

  • Is favored in the absence of substrate.

• The R state:

  • Has higher affinity for substrate.

  • Is the most active form.

  • Is favored upon binding of substrate.

Question: Mutation and ATCase

• A mutation that decreases $K_M$ essentially increases the enzyme's affinity for its substrate, favoring the R state.

Covalent Modification

• Phosphorylation is a common covalent modification.

• Terminal phosphate group from ATP is attached to the hydroxyl groups of Ser, Thr, or Tyr.

• Done by enzyme called kinase.

• Most covalent modifications are reversible.

• Phosphate removed by enzyme called phosphatase.

• Phosphate group alters charge.

Kinase Activation

• Activation of the insulin receptor by phosphorylation.

• The activation loop blocks the substrate-binding site in the inactive state.

• Triply phosphorylated activation loop moves dramatically, making room for the target protein in the substrate-binding site.

Regulation by Phosphorylation of Glycogen Phosphorylase

• Phosphorylase a: phosphorylated (active) form of the enzyme, has a phosphoryl group on Ser 14. Catalyzes bond cleavage.

• Phosphorylase b: the dephosphorylated form.

Isozymes

• Isoenzymes or isozymes are enzymes that are encoded by different genes.

• Isozymes catalyze the same reaction but display different regulatory properties.

• Isozymes may be expressed in a tissue-specific or developmentally specific pattern.

• Example: Glycogen phosphorylase isozymes expressed in muscle, brain, and liver.

Glycogen Phosphorylase a and b Structure and Activity

• Dimer of 842 residue subunits.

• Larger N-terminal domain – glycogen binding subdomain, interface domain, allosteric effector site, Ser-14 phosphorylation site.

• Active site is at the center of the subunit.

• Regulated by allosteric interactions and phosphorylation/dephosphorylation.

• Structures of phosphorylase a and b are similar.

Conformational States of Glycogen Phosphorylase a and b

• T state: surface loop blocks substrate access.

• R state: Ser 14 phosphorylation reorients surface loop allowing substrate access.

• The phosphate recognition site is fully formed only in the R state: Arg 569.

Different Enzymatic forms of Phosphorylase Respond to Different Allosteric Effectors

• Both phosphorylase a and b can be in either the R or T states.

• The equilibrium for phosphorylase a favors the R-state (Active).

• Phosphorylation shifts the equilibrium to the R state.

• The equilibrium for phosphorylase b favors the T-state (Inactive).

In Muscle, Phosphorylase b Activity Affected

• Phosphorylase needs to be active during muscle contraction.

• When a muscle contracts, ATP is converted into AMP.

• AMP (allosteric effector) binds to a nucleotide-binding site and stabilizes the conformation of phosphorylase b in the R state.

• ATP (allosteric inhibitor) competes with AMP and favors the T-state (G6P also favors T-state).

• b: Default state in muscle.

In Liver, Phosphorylase a Responds to Glucose

• Phosphorylase a exhibits the most responsive T-to-R transition (not b form).

• Binding of glucose (allosteric inhibitor) shifts the a form to T state from R state, thus inactivating it.

• High blood glucose level after a meal, there is no need to break down glycogen into glucose.

• Low blood glucose level shifts the equilibrium to the R state to break down glycogen into glucose.

• Unlike in muscle, liver phosphorylase is insensitive to regulation by AMP (recall isozyme definition).

Exam-Style Questions and Answers

• Question 1: How might the (reversible) attachment of an inorganic phosphate group to a hydroxyl-containing amino acid side chain (Ser, Thr, or Tyr) alter the activity of an enzyme?

  • Answer: B. Addition of the phosphate group changes the shape of the enzyme.

• Question 2: Which of the following methods of altering enzyme activity is irreversible?

  • Answer: E. None of the above.