Enzymes Part II

ΔG‡

represents the change in free energy between the reactants and the transition state (X ‡, a species intermediate in structure between S and P) of a chemical reaction, not the products

• Larger ΔG‡ indicates higher energy barrier that must be overcome, resulting in slower reaction

• Smaller ΔG‡ means lower energy barrier and faster reaction

What is essential for catalysis to occur?

tight binding of the transition state is essential

• enzyme must stabilize EX‡ more than it stabilizes ES

• Enzymes bind the transition-state structure more tightly than the substrate product

• The dissociation constant for the transition state (k_T) must be much smaller than for the substrate (K_s)

How Does Destabilization of ES Affect Enzyme Catalysis?

• If the enzyme stabilized the substrate and transition state equally no catalysis would occur

• Favorable interactions provide inartistic binding energy (ΔG_b) to form ES

• Key Concept: If the enzyme stabilized ES and EX‡, ΔG‡ would not change and no catalysis would occur. The enzyme must bind the transition state more tightly than substrate

Mechanisms of Destabilization (ΔG_d)

The free energy of destabilization(ΔG_d) includes strain, distortion, and de-solvation

• Strain/distortion: substrate fit is imperfect bc the active site is designed for the transition state structure → causes strain in the substrate, enzyme, or both

• De-solvation: charged groups on the substrate lose their stabilizing salvation shell when entering the active site → makes charged groups less stable and more reactive

Nucleophile

an electron-rich species that donates a pair of electrons to an electrophile to form a chemical bond

• usually negatively charged or have a lone electron pairs

Biochemical Nucleophiles

• serine (-OH) → serine proteases

• Cysteine (-SH) → Cysteine proteases

• Lysine (-NH₂) → schiff base formation

• Histidine (imidazole group) → act both as nucleophile and general base

• Water (H₂O) → acts as nucleophile in hydrolysis

Covalent Catalysis

the enzyme forms a temporary covalent bond with the substrate, creating a more reactive intermediate that enables the reaction

• increases rate of biochemical reactions by stabilizing high-energy intermediates via covalent bonding

How does covalent catalysis occur:Chymotrypsin Mechanistic Steps

Chymotrypsin cleaves the carboxyl side of large hydrophobic or aromatic amino acids such as Phe, Met, Try, and Trp

1. S binds to E, ES complex formed

2. Nucleophile resides(See, Cys, Lys, His) in active site attack an electrophile center on S, creating a covalent bond

3. Tetrahedral intermediate (EX‡) is formed, the enzyme stabilizes the high energy transition state

4. Product is formed through rearrangement or bond cleavage (EP complex)

5. Water(or another nucleophile) attacks the intermediate to break the EP complex

6. Product is released (E+P)

Acid Base Catalysis

A proton is transferred to catalyze reactions

• Steps: Nucleophilic attack of base, proton transfer(stabilization of transition state), product forms

Acid Base Catalysis Examples

Serine(a) and Aspartic Acid (b) proteases are examples of enzymes that employ general acid-base catalysis

a) In the active site of serine proteases, the serine residue is usually paired with a proton-withdrawing group to promote nucleophilic attack of the peptide

b) Aspartyl proteases activate a water molecule to serve as a nucleophile, rather than using a functional group of the enzyme itself

General vs Specific Acid-Base Catalysis

General acid-base catalysis involves any proton donor or acceptor in the reaction mechanism, while specific acid-base catalysis refers to the use of hydronium or hydroxide ions from the solvent for proton transfer.

• For specific: depends only on pH, rate unchanged at fixed pH, strong acids and bases in solution

Low-Barrier Hydrogen Bonds (LBHBs)

• proton is shared equally between donor and acceptor (not localized on one atom)

• They form when heteroatom distance shrinks to <0.25nm

• Unusually strong: up to ~60kJ/mol (vs. 10-30kJ/mol for typical H-bonds)

• Stabilize transition states → lower activation energy in enzyme catalysis

Low-Barrier Hydrogen Bonds Mechanistic Steps

1. S binds to E via hydrogen bonding (ES-complex formed): Asp, Glu, His, Try are common residues for H-bonding

2. Hydrogen bond strengthening, the hydrogen bond is shortened. Transition state is stabilized

3. Transition State collapses through bond cleavage or formation

4. LBHB reverts to normal hydrogen bond

Metal Ion Catalysis

Metal cofactors orient the substrate and stabilize the transition state

• Mechanism: water activates → substrate binds (TS stabilized) → leaving group departs

• Ex:

  • Thermolysin ZN²⁺ stabilizes neg charge on peptide carbonyl oxygen → Glu deprotonates water → generates -OH → OH attacks carbonyl carbon → peptide bond cleaved

Enzyme Regulation

The mechanisms that control enzyme activity and ensure that metabolic processes are properly coordinated

• Importance: necessary for maintaining homeostasis, controlling metabolic pathways, and responding to environmental changes

Factors that affect enzyme regulation

1. The availability of substrates and cofactors usually determines how fast the reaction goes

2. As product accumulates, a decrease rate of the enzymatic reaction is observed

3. Genetic regulation of enzyme synthesis and decay determines the amount of enzyme present at any moment

4. The presence of allosteric regulators or inhibitors

5. Zymogens, isozymes, and modulator proteins

6. Compartmentalization

Cooperativity

refers to the interaction between different subunits of an enzyme or protein in response to ligand binding, which affects the binding affinity of other subunits

• Not typically observed in monomeric proteins

positive vs negative cooperativity

Positive: substrate binding increases affinity and enzyme activity

Negative: binding decreases affinity, reduces bonding, and flattens the curve

• happens more gradually, helps prevent over activation

Isoenzymes

Different forms of an enzyme that catalyze the same reaction

• differ in amino acid sequence for fine regulation

• Show tissue-specific expression

• arise from gene duplication or alternative splicing

• Regulated by gene expression, allosteric control, and modifications

Allosteric Regulation

Regulation of enzyme activity through binding of an effector molecule at a site other than the active site (allosteric site)

• effectors may be feed-forward(positive) activators or feedback (negative) inhibitors

Sigmoid Curve

A characteristic graph shape representing enzyme activity in allosteric regulation, indicating the relationship between substrate concentration and reaction velocity.

• It shows a slow increase at low substrate levels, followed by a steep rise, and levels off at higher substrate concentrations.

2 Models of allosteric regulation

MWC and KNF

Why doesn’t hemoglobin behave like myoglobin?

• Myoglobin binds O₂ with a hyperbolic curve → simple, single site behavior

• Hemoglobin binds O₂ with a sigmoidal curve - the first O₂ makes binding of the next easier

• Positive cooperativity

MWC conformation election

the protein is already flipping between two states, The lignin doesn’t cause the change - it picks a winner

1. Pre existing equilibrium: The oligomer is flipping between T (taut, low affinity) and R (relaxed, high affinity) even without ligand

2. Symmetry Rule: All subunits must be in the same state. No mixed oligomers are allowed

3. S binds preferentially to R, pulling the equilibrium toward R. The next subunit is already in R ready to go

• Only allows for positive cooperatively

MWC: How activators and inhibitors fit the same logic?

A and I bond elsewhere on the protein and don’t compete with S → they tell the T/R equilibrium before S arrives

• Substrate: binds to R only, pulls equilibrium to R → sigmoidal

• Activator: binds to R only, stabilizes R for S → more hyperbolic

• Inhibitor: binds to T only, stabilizes T so S has a harder time → more sigmoidal

KNF Conformation Induction

ligand biding causes conformational change. The change then spreads - one neighbor at a time

1. Induced fit: S binding deforms the subunit it binds to. No pre-existing R state is required

2. Change transmits to neighbor: the deformed subunit pushes on its neighbor, altering that neighbors affinity for S

3. No symmetry rule: Mixed conformations are allowed, subunits don’t have to match

• Allows for positive and negative cooperativity

MWC or KNF

MWC says ligand picks conformation, KNF says ligand makes it. Reals proteins probably do both

• MWC works great in hemoglobin

• KNF is more flexible and required for negative cooperativity

Zymogens

Inactive enzyme precursors that require activation.

• require proteolytic cleavage for activation

• change is irreversible, once cleaved, zymogens cannot return to inactive state

• enzymes activated only when and where they are needed which prevents premature enzyme activity

Examples of Zymogens

Chymotrypsinogen: Pancreas

Trypsinogen: Pancreas

Procarboxypeptidase: Pancreas

Proelastatse: Pancreas

Pepsinogen: Stomach

Covalent Modification

Enzyme Activity is regulated through the addition or removal of chemical groups (phosphorylation, methylation, acetylation)

• each protein kinase targets specific protein for phosphorylation

• Phosphoprotein phosphates catalyze the reverse reaction - removing phosphorylation groups from proteins

Covalent modification Example

• Example: Glycogen phosphorylase activated by phosphorylation

• Reversible Modification: phosphorylation/dephophorylation by kinases and phosphates

Regulating Enzymes via Covalent Modification

• Kinases and phosphatases are targets of regulation

• protein kinases phosphorylate Ser, Thr, and Try residues in target proteins

• All Kinases share a common catalytic mechanism based on a conserved core kinase domain of about 260 residues

Cyclic AMP-Deoendent Protein Kinase

Cyclic AMP-Dependent protein Kinases (PKA): catalytic(C) and regulatory(R) subunits

• PKA is a tetramer in mammalian cells

• The two R subunits bind to equivalents of cAMP each; binding relates the R subunits from C subunits

• C subunits are enzymatically active as monomers

Glycogen Phosphorylase(GP)

dimer of identical 842 residue subunits, each containing an active site and an allosteric effector site near the subunit interface

• GP cleaves glucose units from nonreducing ends of glycogen through phosphorolysis reaction to convert to usable cellular fuel

Regulatory Phosphorylation Site

Ser14 on each subunit. An allosteric effector site also exerts

regulatory control

Glycogen Phosphorylase Steps

1. Acid Catalysis: PLP’s 5’-phospagte protonates Pi, which then protonates glycosidic O, weakening C-O bond

2. Glycosidic bond cleaves; transient carbocation forms at C1. Glycogen shortens to n-1 residues

3. Nucleophilic Attack: Activated Pi attacks C1, releasing α- D-glucose-1-phosphate.

How does allosteric control regulate Glycogen Phosphorylase (GP) under the MWC model? (Include states, activators, inhibitors, and the "bottom line" speed/reversibility).

• Conformational States: R = active; T = inactive.

• Activator: AMP drives the shift from inactive to active (T→R).

• Inhibitors: ATP, G6P, and caffeine drive the shift from active to inactive (R→T).

• Mechanism: Involves a conformational change at the protein subunits.

• Bottom Line: Provides fast, reversible control.

How does covalent control regulate Glycogen Phosphorylase (GP)? (Include the b vs. a forms, the structural modifier, the cellular trigger, and the "bottom line" effect).

• Forms:

  • Phosphorylase b = less active, allosteric-dependent.

  • Phosphorylase a = phosphorylated, fully active.

• Enzymes Involved: Phosphorylase kinase adds the phosphate (b→a); Phosphoprotein phosphatase 1 removes it (a→b).

• Physiological Trigger: High ATP demand (which completely overrides allosteric control).

• Bottom Line: Provides sustained activation via covalent modification (phosphorylation).

How do cellular energy levels allosterically regulate Glycogen Phosphorylase (GP) activity? Describe the metabolic conditions, the effectors, and the resulting kinetic curve changes (T vs. R state).

• High Energy State (Inhibition):

  • Condition/Effectors: Abundant ATP and G-6-P (signals high energy reserves).

  • Pathway Effect: Glycogen breakdown is inhibited.

  • Kinetics/Curve: Favors the T state; the curve becomes more sigmoidal (shifts to the right).

• Low Energy State (Activation):

  • Condition/Effectors: High [AMP] / Low [ATP] and [G-6-P] (signals low energy reserves).

  • Pathway Effect: Glycogen catabolism is stimulated.

  • Kinetics/Curve: Favors the R state; the curve becomes more hyperbolic (shifts to the left).

• Key Detail: AMP and ATP compete because AMP binds at the same allosteric site as ATP.

Regulation of GP by covalent modification

Hormone regulated enzymatic cascade leads to conversion of GP b(inactive) to GP a(active) form

Adenylyl Cyclase Reaction

The reaction is driven forward by subsequent hydrolysis of pyrophosphate by enzyme inorganic pyrophosphatase

cAMP as a second messenger

cAMP is the intracellular agent of extracellular hormones - thus a second messenger

• hormone bonding stimulates a GTP-binding heterotrimeric protein, triggering release of its Gα(GTP)

Genetic Regulation of Enzyme Levels

transcriptional Control: gene expression can be upregulated or down regulated in response to cellular needs

• Example: Lac operon in prokaryotes

• Presence of lactose up regulates the expression of lacZ and lacY, and lacA genes

• Absence down regulates gene expression

Feedback Inhibition

A product of a metabolic pathway inhibits and earlier step in the pathway to regulate the flux of intermediates

• mechanism: prevents overproduction of end products

Regulation by compartmentalization

Enzymes, substrates and regulatory molecules are located in separate compartments so that opposing pathways are physically separated, or located close together, to increase pathway efficiency

• enzymes may also be attached to the cytoskeleton, or to a membrane