BIOMG 1350 Lecture 5 - Proteins in Action

Importance of genetic code redundancy

• The genetic code is redundant and multiple codons encode for the same amino acid

• There are 3 stop codons that are similar (UAA, UAG, UGA) so that a mutation will result in a stop codon as well and preserve the genetic code's meaning

• Codons encoding for similar amino acids (such as hydrophobic ones) tend to be similar to one another too.

Protein binding and interactions with molecules

3D structure allows proteins to execute certain functions

Selective binding of a protein to a ligand depends on a set of weak noncovalent interactions (h bonds, van der waals, electrostatic, and hydrophobic interactions) and binding sites in proteins interact with very specific ligands

Folding into the 3D protein structure ideally positions critical sidechains in a way that allows for very specific non covalent interactions with the ligands the protein needs to interact with

Enzymes, a type of protein, are a key molecular machine for proteins to interact with molecules. Proteins function by making specific interactions with other molecules

Proteins can interact with other proteins and ligands which involve many weak noncovalent interactions (electrostatic interactions, hydrogen bonding, van der waals, and hydrophobic)

Ligand

often a substance (ion, small organic molecule, or macromolecule) that is bound by a protein

This binding typically occurs at a specific site (like a receptor's binding pocket) and can trigger a biological response, such as activating a signaling pathway or changing the shape/function of the target protein.

Common examples include hormones (like insulin binding to its receptor), neurotransmitters (like dopamine binding to dopamine receptors), and substrates binding to enzymes.

Spontaneous Process

A process that occurs naturally without added energy, often resulting in an increase in entropy or a release of free energy.

Entropy

A measure of disorder or randomness in a system; as energy is transformed, entropy tends to increase, meaning systems naturally move toward more disorder.

Ex: During cellular respiration, glucose is broken down into CO₂ and H₂O. The overall disorder of the universe increases because energy is released as heat and the products are more random. However, cells maintain local order by using some of that energy to build and organize molecules like proteins and DNA.

Gibbs Free Energy

The portion of a system’s energy that can be used to do work at constant temperature and pressure; changes in Gibbs free energy (ΔG) determine whether a process is spontaneous (ΔG < 0) or non-spontaneous (ΔG > 0).

Equation is ΔG=ΔH−TΔS where:

• ΔG = change in free energy (spontaneous if negative)

• ΔH = change in enthalpy (total energy)

• T = temperature in Kelvin

• ΔS = change in entropy

Endergonic Reaction and Exergonic Reaction

Ender: A chemical reaction that absorbs free energy from the surroundings, has a positive ΔG, and is non-spontaneous (requires energy input to occur, e.g., photosynthesis).

Exer: A chemical reaction that releases free energy to the surroundings, has a negative ΔG, and is spontaneous (e.g., cellular respiration).

Gibbs Free Energy and Natural Decrease

In any system, Gibbs free energy (G) naturally decreases as reactions proceed toward equilibrium (or higher entropy), meaning energy is released and reactions tend to be spontaneous (ΔG < 0). To increase free energy or build order (ΔG > 0), energy input is required—for example, cells use ATP to drive endergonic reactions like synthesizing macromolecules.

Key idea: Systems naturally move toward lower free energy and more stability, but living organisms maintain order by constantly adding energy.

Energy Coupling

The use of energy released from an exergonic reaction (ΔG < 0) to drive an endergonic reaction (ΔG > 0), allowing cells to perform work efficiently (e.g., ATP hydrolysis powering the synthesis of macromolecules).

ATP (Adenosine Triphosphate)

The primary energy currency of the cell, storing potential energy in its high-energy phosphate bonds; energy is released when ATP is hydrolyzed to ADP and Pi (i stands for inorganic) to power cellular work.

ATP is the most widely used chemical energy source in cells since ATP hydrolysis releases a lot of energy.

• The energy released by ATP hydrolysis is coupled to hundreds of reactions in cells

• ATP + H2O -> ADP + Pi

• ATP is regenerated from ADP by energy from sunlight or food

Phosphorylated Intermediate

A molecule temporarily bonded to a phosphate group from ATP, which raises its energy and makes it more reactive, allowing it to drive an endergonic reaction.

Key idea: It’s the direct link in energy coupling—ATP donates a phosphate, creating the intermediate that performs the work.

Energy Coupling via ATP Hydrolysis

• ATP Hydrolysis:

  • ATP is hydrolyzed to ADP + Pi.

  • The released phosphate (Pi) carries energy that can be transferred to another molecule.

• Formation of a Phosphorylated Intermediate:

  • The Pi from ATP covalently attaches to a target molecule, forming a phosphorylated intermediate.

  • This attachment raises the energy of the molecule, making it more reactive.

• Reaction Occurs:

  • The phosphorylated intermediate can now undergo the endergonic reaction it previously couldn’t.

  • For example, it might bind to a substrate or change shape to drive chemical, transport, or mechanical work.

• Release and Reset:

  • After the reaction, the phosphate is released and the two reactant molecules are combined (like Glutamic acid and Ammonia).

  • ATP is regenerated later in metabolism to repeat the cycle.

Previously the gibbs free energy of the total reaction (Glutamic acid + Ammonia → Glutamine, for example) was positive. The energy released by the ATP and attached to one of the reactants causes the total gibbs free energy of the system with ATP to be negative meaning although the combination of Glutamic Acid and Ammonia is positive (not spontaneous) the overall entropy of the system increases with a net negative gibbs free energy.

Regeneration of ATP

The process of reforming ATP from ADP + Pi using energy from cellular processes so cells can continuously perform work. ATP + H2O results in energy (for cellular work) + ADP + Inorganic Phosphate. ADP + Pi is converted back into ATP via energy from catabolism (exergonic, energy releasing processes like breakdown of glucose).

How it Happens:

1. Substrate-Level Phosphorylation: Direct transfer of a phosphate from a high-energy molecule to ADP (e.g., glycolysis).

2. Oxidative Phosphorylation: Energy from the electron transport chain creates a proton gradient that drives ATP synthase to make ATP (cellular respiration).

3. Photophosphorylation: In photosynthesis, light energy generates a proton gradient in chloroplasts that drives ATP formation.

Reversable Process.

Activation energy

The amount of energy that reactants must absorb before a chemical reaction will start; also called free energy of activation.

It represents the energy needed to break or weaken existing chemical bonds so that new bonds can form. Enzymes lower activation energy by stabilizing the transition state, allowing reactions to occur faster without being consumed or changing the overall energy of the reaction.

Enzyme

A biological catalyst, usually a protein, that speeds up chemical reactions by lowering activation energy without being consumed; it is specific to its substrate due to the shape and properties of its active site.

Substrate

The specific reactant molecule that binds to an enzyme’s active site and is acted upon during an enzyme-catalyzed reaction.

Active site

The region of an enzyme where the substrate binds and the chemical reaction is catalyzed; its shape and chemical properties determine enzyme specificity.

Catalysis

The process by which a substance (a catalyst, such as an enzyme) speeds up a chemical reaction by lowering activation energy without being consumed or permanently changed.

Enzyme–substrate complex

The temporary structure formed when a substrate binds to an enzyme’s active site during a catalyzed reaction.

Effects of Temperature and pH on Enzyme Activity

Enzyme activity is influenced by environmental conditions:

Temperature: Increasing temperature generally speeds up reactions by increasing molecular collisions, but extreme heat can denature the enzyme, altering its shape and destroying activity. Low temperatures slow enzyme activity.

pH: Each enzyme has an optimal pH range; deviations can disrupt ionic and hydrogen bonds, changing the enzyme’s shape and reducing or stopping activity. Extreme pH levels can denature enzymes.

Generally enzymes are at their optimal ability, or Vmax, or highest rate of reaction when they are in the pH or Temp they are usually found in. Enzymes in the stomach work best, for example, when they are in the low pH, body temp area of the stomach.

Enzyme denaturation

The loss of an enzyme’s three-dimensional structure due to extreme temperature, pH, or chemical exposure, which disrupts the active site and prevents the enzyme from functioning. Note: denaturation only happens on quaternary and/or tertiary level of enzyme/protein structure as those are the structures held together by IMFs.

Cofactors and Coenzyme

Factors: Any nonprotein molecule or ion that is required for the proper functioning of an enzyme. Cofactors can be permanently bound to the active site or may bind loosely and reversibly, along with the substrate, during catalysis. Essential for its activity, helping the enzyme function properly by stabilizing its structure or participating in the chemical reaction.

Enzymes: A type of organic cofactor, often a vitamin-derived molecule, that binds to an enzyme and assists in catalyzing a reaction by temporarily carrying chemical groups or electrons. Like cofactors they can be permanently bound to the active site or may bind loosely and reversibly, along with the substrate, during catalysis.

Competitive inhibitor

A molecule that resembles the enzyme’s substrate and binds to the active site, blocking the substrate from binding and thus decreasing the rate of the reaction; its effect can often be overcome by increasing substrate concentration. Reduces the activity of an enzyme by entering the active site in place of the substrate, whose structure it mimics.

Ex: Aspirin: Competes with substrate for the active site of cyclooxygenase (COX) enzymes, blocking prostaglandin production and reducing pain and inflammation.

Noncompetitive inhibitor

A molecule that binds to an enzyme at a site other than the active site (an allosteric site), changing the enzyme’s shape and reducing its activity so that the active site no longer effectively catalyzes the conversion of substrate to product., regardless of substrate concentration.

Ex: Sarin (nerve gas): Binds to acetylcholinesterase at a site other than the active site, changing its shape and preventing breakdown of acetylcholine, causing toxic buildup.

Allosteric regulation

The regulation of an enzyme’s activity by the binding of a molecule (activator or inhibitor) to a site other than the active site (the allosteric site), which changes the enzyme’s shape and alters its activity.

Activator: Stabilizes the active form of the enzyme, increasing activity.

Inhibitor: Stabilizes the inactive form of the enzyme, decreasing activity.

Noncompetitive inhibitors are an exmaple of allosteric regulation. However, competitive inhibitors are not.

Allosteric Activation: A regulatory mechanism in which a molecule (allosteric activator) binds to a regulatory/allosteric site (often at the interface of subunits) of a multisubunit enzyme, stabilizing the active conformation of the enzyme. This enhances the activity of all active sites, increasing substrate binding and catalysis. Cooperativity can amplify this effect: binding of a substrate to one subunit can activate all subunits.

Allosteric Inhibition: A regulatory mechanism in which a molecule (allosteric inhibitor) binds to a regulatory/allosteric site of a multisubunit enzyme, stabilizing the inactive conformation of the enzyme. This decreases the activity of all active sites, reducing substrate binding and catalysis.

Feedback Inhibition

A regulatory mechanism in which the end product of a metabolic pathway binds allosterically to an enzyme that acts early in the pathway, inhibiting its activity. This prevents the overproduction of the product and conserves cellular resources.

Example: In the synthesis of isoleucine from threonine (which involves multiple intermediates), accumulated isoleucine allosterically inhibits the first enzyme in the pathway, slowing its own production. This way if there is too much isoleucine production, then production is slowed. But if there is too little, the enzyme that catalyzes the isoleucine reaction production is no longer inhibited and free to work.

Carbohydrates

Monomers of carbohydrates are monosaccharides and polymers are polysaccharides

Carbohydrate contains carbon + water (some variation of (CH2O)n)

Like DNA and RNA sugars can form long polymers by condensation reactions which form glycosidic bonds (covalent bonds)

Monosaccharides (1 carbohydrate monomer), disaccharides (2 carbohydrate monomers connected), oligosaccharides (3-10 carbohydrate monomers connected), and polysaccharides (>10 carbohydrate monomers connected)

Polysaccharides are built via condensation reactions and reduced back to their monomer form or broken up via hydrolysis

Properties of different carbohydrates

Polysaccharides are important for energy storage (glycogen), structural support (cellulose in plants, extracellular matrix in animals, and cell walls in bacteria and fungi)

Glycogen is an example of a branched polysaccharide (around 50,000 glucose monomers). Polysaccharides can be branched unlike RNA and DNA.

Protein and lipid modification is also a function of polysaccharides

Cellulose is the most abundant macromolecule and about 1/3 of plant mass is cellulose

If hydrolysis is energetically favorable, why are macromolecules like DNA stable in cells? Shouldn’t they all fall apart?

Activation energy keeps macromolecules stable and prevents spontaneous hydrolysis. Because even with hydrolysis, and the fact its energetically favorable, it still needs to get over that activation energy hump where an enzyme is needed.

How are polysacchardies broken apart?

Lysozymes can cleave polysaccharides, and lowers the activation energy allows cells to regulate processes

Lysozyme is the specific enzyme that helps to hydrolyze the polysaccharides in bacterial cell walls. They serve as the enzyme and the polysaccharide is the substrate which forms an enzyme substrate complex with the lysozyme and then the polysaccharide is hydrolyzed.

Although surrounded by water, polysaccharides do not undergo hydrolysis without lysozymes. The activation energy is too high for the reaction to occur spontaneously even though it is favorable.

How do enzymes function and lower the activation energy of a reaction?

Enzymes can lower the activation energy by binding to their substrate and bringing the substrates together in proper orientation for the reaction to occur, altering the distribution of electrons in the substrates, and change the shape of the substrates (bending certain bonds).

Enzymes can only catalyze favorable reactions which means an overall reduction in energy state

Energy mechanisms can involve the transient formation and subsequent breakage of covalent bonds to lower the activation energy for a reaction.

Enzymes catalyze chemical reactions while remaining unchanged themselves

Uncatalyzed reaction occurs very slowly or basically not at all, but catalyzed reactions occur much faster.

Process of enzymes catalyzing reactions

Substrates bind to the active site of enzymes. Then an enzyme substrate complex is formed, and then the enzyme catalyzes a chemical reaction that its substrate can undergo. Then the substrate product, now changed from the enzyme's reaction, is released from the enzyme substrate complex, and the enzyme can perform another reaction catalysis.

Enzymes speed up reactions by lowering the activation energy barrier. This is for spontaneous reactions or reactions that increase in entropy.

Enzyme are necessary as the activation energy barrier must be lowered for the molecule/substrate to change or get over the transition state. Otherwise the substrate/molecule will basically never transform.

Lock and key validity for modeling enzyme function

Lock in key is an incorrect model for enzyme and substrate interactions. In reality, enzymes break and bend the key to make it conform into the "lock" for manipulation.

Reaction Coupling

Reacting coupling can drive unfavorable reactions.

One can understand reaction coupling through an example: If reaction 1 is unfavorable and will not occur naturally and requires an input of 23 kJ/mol of energy, and reaction 2 is favorable and will occur naturally and will release 30.5kJ/mol, reaction 2's excess energy (more than the energy needed for reaction 1) can be used to drive reaction 1 despite it being unfavorable.

For coupled reactions the total energy in the reaction must be lost which aligns with the fact that entropy must always increase overall despite it decreasing in the instance of 1 reaction.

Hydrolysis of ATP into ADP and P is usually the favorable reaction that is driving the unfavorable reactions in the cell. This is the reaction that is coupled with other unfavorable ones.

The question of enzymes being used for theromodynamically unfavorable reactions

Enzymes speed up reaction rates, but do not change whether a reaction is favorable or not. Essentially, enzymes cannot turn an energetically favorable reaction into an energetically unfavorable reaction or vice versa.

Enzymes cannot change the relative energies of the substrates and products.

Then the question becomes how do cells synthesize all these polymeric macromolecules, if a condensation reaction is unfavorable? The answer is that enzymes can couple favorable and unfavorable reactions such that an unfavorable reaction could occur if the overall system (combination of both favorable and unfavorable) has a net loss of energy or is overall favorable.

Different ways protein activity can be regulated and protein amount can be controlled

There are multiple different ways of how protein activity is regulated in cells but they are mainly regulated through abundance and activity

• They can control the protein amount

  • Rate of mRNA transcription (gene expression)

  • Rate of mRNA degradation

  • Rate of mRNA translation into protein

  • Rate of protein degradation

• Control of protein activity

  • Cellular localization (targeting to nucleus)

  • Inhibition or activation by ligands (feedback inhibition)

  • Inhibition or activation by another protein

  • Protein modification (phosphorylation)

Protein phosphorylation

Phosphorylation can regulate protein activity

• Proteins are often regulated by reversible phosphorylation of –OH groups on the amino acids serine, threonine or tyrosine. Protein phosphorylation is mediated by kinases, enzymes that transfer a phosphate from ATP to an amino acid side-chain on specific proteins.

• Phosphorylation is a very common way of regulating protein function and this changes how the protein interacts with other molecules.

• A substrate protein can be phosphorylated by kinase which is the process of adding a phosphate group onto the substrate protein. Then, the phosphorylated substrate protein can perform a different function and is "activated."

• A protein phosphatase can take off the phosphate group from the phosphorylated protein to "deactivate" it or turn it off.

Prevalence of protein regulation by phosphorylation

Regulation by phosphorylation is very common and there are about 518 protein kinases in humans (or 2% of the proteome (the entire set of proteins expressed by the genome)).

How does phosphorylation of a protein affect its activity?

Phosphorylation could increase or decrease activity. In other words, a protein can either become activated or inactivated by phosphorylation and isn’t always turned “on” by phosphorylation.

Protein Kinase

An enzyme that transfers a phosphate group from ATP to a specific amino acid (usually serine, threonine, or tyrosine) on a target protein, a process called phosphorylation. This modification often changes the target protein's activity, function, or interactions, making kinases key regulators of cell signaling pathways.

• Each protein kinase has one or many substrate (target) proteins.

• All kinases share a common kinase domain, but with differences to make them highly specific for specific substrates

• Kinases often phosphorylate other kinases in signaling pathways

Protein Phosphatase

An enzyme that removes a phosphate group from a protein, a process called dephosphorylation, effectively reversing the action of a kinase. By counteracting kinases, phosphatases help control the "on/off" switching of signaling pathways and maintain the balance of phosphorylation within the cell.

• There are around 200 phosphatases in humans with over 51,000 phosphorylation sites in the human proteome

Small GTP-binding protein and their regulation pathway

Small GTP binding protein is a signaling protein that acts as a molecular switch by switching between an inactive GDP-bound state and an active GTP-bound state. When GTP is bound, the protein is active and can transmit signals to other proteins.

• GEF stands for guanine nucleotide exchange factors and these switch out GDP for GTP on the small GTP binding proteins usually turning the protein "on"

• GAP stands for GTPase activating protein and these promote the removal of the phosphate group from GTP which eventually results in the small GTP binding protein to be turned "off"

• Once the small GTP binding protein has a GTP attached and is in the "on" state, it can bind to "effector" proteins to activate a downstream pathway. Examples of effectors include protein kinases. By binding an effector, they activate it.

• The rates of GTP binding and GTP hydrolysis for small GTPases are controlled by other proteins.

• How does binding of GTP to a GTP binding protein affect its activity? GTP binding to a GTP binding protein alwaysactivatesthe protein.

GEF (Guanine nucleotide Exchange Factor)

A regulatory protein that activates a small GTP-binding protein by helping it release GDP so that GTP can bind. In other words, GEF switches the protein from the GDP-bound “OFF” state to the GTP-bound “ON” state.

GAP (GTPase-Activating Protein)

A regulatory protein that helps turn off a small GTP-binding protein by stimulating the hydrolysis of GTP into GDP. This causes the GTP-binding protein to return to its inactive GDP-bound state.

Prevalence of protein regulation by GTPases (or small GTP binding proteins)

There are over 100 small GTPase proteins (a.k.a. small GTP binding proteins) in the Human genome and each small GTPase (small GTP binding protein) regulates a downstream pathway from membrane traffic to regulation of the cytoskeleton to growth control.

RAS and malfunction of the small GTP binding protein activation cycle

An oncogene (mutated gene that has the potential to cause cancer) was the first small GTPase (small GTP binding protein) to be identified. When GEF swaps out GDP for GTP on RAS, the RAS small GTP binding protein activates and it sends out a cell growth signal.

RAS G12V mutation leads to cancer as the RAS protein cannot hydrolyze GTP and thus RAS is always active and propagating cell growth signals.

Effector proteins

Effector proteins are proteins that receive a signal from an activated signaling protein and then carry out the next step in the signaling pathway.

Rather than relaying the signal further, effectors directly produce the cell's response—for example, an enzyme that synthesizes a second messenger, or an ion channel that opens to let ions flow across the membrane. A classic example is adenylyl cyclase, which is activated by a G protein and produces cAMP, triggering downstream effects in the cell.