Reaction Coupling and Metabolic Pathways

Lecture 2: Part I - Reaction Coupling

• Reaction coupling combines unfavorable and favorable reactions.
• Understanding reaction coupling involves free energy diagrams.
• Activation energy is key in enzyme reactions.
• Free energy diagrams can illustrate coupled reactions.

Classes of Metabolic Pathways

• Metabolic pathways are either catabolic or anabolic.
• Catabolism involves breakdown, releasing free energy.
• Anabolism involves construction/synthesis, consuming energy.
• Catabolic reaction pathways are exergonic.
• Anabolic reaction pathways are endergonic.

Favorable vs. Unfavorable Reactions

• A favorable reaction/process is catabolic, produces energy, and has a negative ΔG.
• An unfavorable reaction/process is anabolic, requires energy, and has a positive ΔG.
• ΔG represents the difference in energy levels between reactants and products.
• Endergonic reactions enter energy and require energy, resulting in being non-spontaneous.
• Exergonic reactions exit energy and proceed spontaneously, releasing energy.

Coupling Reactions

• Coupling reactions involve both endergonic and exergonic processes.
• A mechanical example involves work done raising an object (endergonic) coupled with the loss of potential energy from the object's position (exergonic).
• Chemical examples require chemical and electrical energy.

Activation Energy

• Activation energy is always positive.
• Activation energy is required to start a reaction.

Reaction Coupling Example

• Reaction 1 (Glucose + Pi → glucose 6-phosphate) is endergonic.
• Reaction 2 (ATP → ADP + Pi) is exergonic.
• Reaction 3 (Glucose + ATP → glucose 6-phosphate + ADP) involves coupling of the previous two reactions.
• ΔG3 = ΔG1 + ΔG2 in this example.

Metabolic Coupling Reactions

• Oxidation-reduction reactions always occur together.
• Oxidation-reduction reactions play a key role in biological energy flow.
• Electrons carrying energy are transferred between molecules.
• Energy production during cellular respiration and photosynthesis relies entirely on oxidation-reduction reactions.
• NAD+ is a carrier of H+ and electrons.
• NAD+ is nicotinamide adenine dinucleotide.
• NAD+ is an oxidizing agent.
• NAD+ accepts electrons and gets reduced.
• NADH is a reducing agent
• NADH donates electrons and gets oxidized.
• NADH carries 2 electrons and 2 protons.

Lecture 2: Part II - Enzyme Kinetics

• Enzymes play a critical role in biochemical reactions.
• Reading equilibrium diagrams is important for reactions with and without enzymes.
• Enzymes affect the equilibrium and processes of reactions.
• Enzymes affect free energy via free energy diagrams.
• Velocity and rate constants are related.
• Enzyme binding sites are important.
• Rate constants and equilibrium constants are different.
• Products vs time plots are important in respect to steady state enzyme kinetics.
• Michaelis-Menten plots can be produced and read.
• Lineweaver-Burke plots can be produced and read.
• Vmax and Km can be estimated from Michaelis-Menten and calculated from Lineweaver-Burke plots.

Enzymes

• Enzymes catalyze reactions by increasing the rate without being changed themselves.
• Enzymes are highly specific and recyclable.
• All enzymes are proteins, but not all proteins are enzymes.

Equilibrium

• Equilibrium is reached when the concentrations of reactants and products no longer change.
• At equilibrium, forward and reverse rates are equal, and concentrations stay constant until the equilibrium is disturbed.

Equilibrium with Enzymes

• Enzymes speed up the rate of reaction.
• Enzyme do not alter the equilibrium end-point.
• With enzymes, equilibrium is reached faster.
• .Enzymes lower the activation energy, but do not change the ΔG.

Enzyme Specificity

• Enzymes are specific for each reaction.
• Specificity is related to size, shape, electrostatic properties, hydrophobicity, and hydrophilicity.

Stereospecificity of Enzymes

• Isomers have the same chemical formula but different spatial arrangement of the atoms.
• Enantiomers are mirror-image molecules and are structurally equivalent but not superimposable.
• Enzymes are also stereospecific.
• If an enzyme demonstrates stereospecificity, it can have negative results with drug use, such as Thalidomide causing birth defects.

Classification

• Oxidoreductases: catalyze oxidation-reduction reactions (BH2 + A ⇄ B + AH2).
• Transferases: catalyze transfer of functional groups from one molecule to another (D-B + A-H ⇄ D-H + A-B).
• Hydrolases: catalyze hydrolytic cleavage (A-B + H2O → A-H + B-OH).
• Ligases/Synthetases: catalyze bond formation, coupled to ATP hydrolysis.
• Lyases: catalyze group removal/addition to double bonds or other cleavages with electron rearrangement (A-B ⇄ A + B).
• Isomerases: catalyze intramolecular rearrangement (R-A-B ⇄ A-B-R).

Rate Constant vs. Equilibrium Constant

• Rate constant (k) is the rate of a reaction involving conversion of reactants into products, measured in moles per second.
• Equilibrium constant (Keq) is the ratio of product concentration to reactant concentration.
• Reaction rate is the amount of reactant converted into product within a specific time period.
• A is the reactant being converted to B
• Velocity = k.[S]
• k is rate constant (k)
• [S] denotes concentration of B
• [A] denotes concentration of A
• At equilibrium: k+1 = K-1

Two-Step Enzyme Reactions

• Kcat represents the number of substrate molecules converted to a product over a specified time for one enzyme.
• k2 is the rate limiting step.
• Concentration of the enzyme substrate complex is constant, i.e. steady state kinetics.
• No reverse reaction (k-2)=0

Steady State Enzyme Kinetics

• Steady state is the state of reaction where the concentration of enzyme-substrate complex is constant.
• Enzymes docking with the linear section of a graph.
• Steady state has a linear phase.
• Steady state is lost during the plateau phase, when no substrate are remain to make a product.

Michaelis-Menten Equation

• Leonor Michaelis and Maud Menten developed an alternate graph to describe enzyme kinetics for a single substrate reaction.
• The components include Vo=Initial Velocity, Vmax=Maximum Velocity, [S]= Substrate concentration, Km=Michaelis constant.

Michaelis-Menten (MM) Plot

• Key features include Vmax=Maximum velocity, Km= Michaelis-Menten constant, and plotting the axis of reaction velocity vs substrate concentration. Km= Michaelis-Menten Constant.
• the substrate concentration equals half Vmax.
• The MM equation is only relevant when the plot shows a hyperbolic relationship between [S] and Vo.

Maximal Velocity (Vmax)

• Vmax is the rate of the reaction when substrate molecules completely fill (saturate) the enzyme's active sites. Reflects how fast the enzyme can catalyze the reaction. Given by the asymptote. As the substrate concentration is extrapolated to infinity. Left and middle image are linear section in graph. Plateau is due to too much substrate and not enough enzymes. • Active sites vacant -> Enzyme can work faster -> Rate can increase. Active sites occupied -> Enzyme cannot work faster -> Rate cannot increase.

High vs Low Km

• High Km: the substrate concentration is much slower.
• Low Km: the substrate concentration is faster.

Lineweaver-Burk equation

• Is divided by Michealis-Menten.
• Includes straight line equations.

Lineweaver-Burk Plot

• Slope is equal to Km over Vomax

Lecture 2: Part III - Enzyme Inhibition

Enzyme Inhibition

• Enzyme Inhibition plots.
• Direct reaction plots.

Enzyme Inhibitors

• Decreases the activity by binding to the enzyme
• There are inhibitors that bind reversibly or irreversibly
• Competitive inhibitor, non-competitive inhibitor, or uncompetitive inhibitor .An enzyme inhibitor is a compound that binds to the enzyme and decreases its activity. Decrease in activity can be caused by the inhibitor preventing the substrate from binding or preventing the enzyme from catalysing thereaction. Inhibitors binding can be reversible or irreversible.

Reversible Inhibitors

• Include Competitive inhibitors that bind to enzyme active site
• Substrate cannot bind
• There's no product to be farmed
• resemble substrate

Non-Competitive Inhibitors

• Non-competitive inhibitors bind to another site on the enzyme(allosteric sites

Un-Competitive Inhibitors

• Uncompetitive inhibitors bind to another site on the enzyme (like non-competitive inhibitors).
• Can only bind to the substrate complex. When the active site is occupied by the substrate i.e. only binds to. Product cannot be be farmed.

Out Competing inhibition

• competitive inhibitors can be overcome by increasing substrate inhibitor

Non- Outcompeting

• None- competitive inhibitors can not be overcome by increasing substrate Inhibitors

Explore reaction coupling, where unfavorable reactions link with favorable ones, illustrated by free energy diagrams. Learn about catabolic and anabolic metabolic pathways, including exergonic and endergonic reactions. Understand favorable and unfavorable reactions based on their energy requirements and ΔG values.