Biochemistry Module I Lesson 2: Enzymes

What is another type of enzyme inhibition that is different than the normal types of inhibition: competitive; no-competitive; uncompetitive.

"Suicide" inhibitors covalently bind the enzyme and prevent it from catalyzing reactions. Since they form covalent linkages to the proteins, they rarely unbind (permanent bonds), which is why they are called suicide inhibitors.

What are some proteins that are non-enzymatic proteins?

- Receptors - ion channels - transport proteins - motor proteins - a special class called antibodies

There are three major families in the CAM family. What are cadherins?

- a group of glycoproteins that mediate calcium dependent cell adhesion. - hold similar cell types together - different types of cells have different cadherins

There are three major families in the CAM family. What are integrins?

- a group of proteins that all have two membrane-spanning chains called alpha and beta. - both chains are important for binding and communicating with extracellular matrix - role in cell signaling (can impact cell function by promoting cell division, apoptosis or other processes)

What is Keratins?

- an intermediate filament protein in epithelial cells - contribute to mechanical integrity to cell - regulatory proteins - makes up hair and nails

Myosin is the primary motor protein located in the myofibril. What other function does myosin posess?

- cellular transport - muscle contraction - movement of neck results in the powerstroke

What is elastin?

- important to extracellular matrix of connective tissues - primary role to stretch and recoil like a spring - restores original shape.

What is tubulin?

- makes up microtubules (provide structure, chromosome separation, intracellular transport with dynein and kinesin) - has polarity - positive end in periphery of cell - negative end is adjacent to the nucleus.

What are motor proteins?

- possess enzymatic activity (ATPase) - transient interactions with actin or microtubules - myosin is the primary motor protein; interacts with actin

What is actin?

- protein making up microfilaments and thin filaments in myofibrils - abundant in eukaryotic cells - positive and negative side; - polarity allows them to travel unidirectionally like a one way street on an actin filament

There are three major families in the CAM family. What are selectins?

- unique proteins that bind to carbohydrate molecules that project from other cell surfaces. (weakest bonds of all CAMs) - role in host defense (inflammation and white cell migration)

What makes phosphorylation such an effective and convenient means of regulation? Hint: 7 Reasons!

1. Net negative charge The neutral residue is transformed into a new negative residue to form new stable interactions - Phosphorylation gives the enzyme two extra negative charges which can disrupt old interactions and form new interactions. This can in turn modify the active site and alter the catalytic rate of the enzyme. 2. Hydrogen Bond Properties - As a result of the negative charge on the residue gives the residue a high potential to form hydrogen bonds with other molecules like the target substrate molecule - Can increase the specificity of the interaction between the active site and the substrate. 3. Kinetics is adjustable by protein kinases - The speed at which phosphorylation occurs can be easily controlled by the cell. The physiological needs of the cell can direct the phosphorylation rate. 4. Abundance of ATP - Phosphorylation requires the transfer of a phosphoryl group from a high energy molecule onto the protein residue. The high abundance of ATP allows the cells to utilize them quickly and effectively in phosphorylation. 5. Amplification effects with protein kinases - Activated protein kinases can be used to regulate many enzymes and reaction pathways all at once. This can lead to amplification effects within the cell. 6. Release Large amount of free energy - dephosphorylation of ATP is an exergonic reaction and it releases a large amount of free energy. - Phosphorylation is thermodynamically favorable and occurs in one direction - products have lower energy than reactants 7. Phosphorylation is reversible - protein phosphatases are enzymes that reverse the effects of protein kinases - using a hydrolysis reaction [CH3CH2OPO3^2- + H2O yields EtOH + HOPO3^2-]

When an antibody binds to an antigen 1 of 3 things may occur...

1. Neutralizing the antigen, making pathogen unable to effect the body. 2. Mark the pathogen for destruction, also called opsonization. Immediately degraded by white blood cells. 3. Agglutinating (clump together) the antigen and antibody in a large insoluble protein complex that can be phagocytized by macrophages.

What are the 6 categories of enzymes?

1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases 5. Isomerases 6. Ligases

What are the 5 steps in an enzyme catalyzing a reaction?

1. The Enzyme and Substrate are free reactants 2. The enzyme and the substrate bind together to form the enzyme substrate complex. 3. This is the binding of the induced fit model. We are at the transition state of the reaction (point of the highest energy). This is where the enzyme is most tightly bound to its substrate. [E-X] (shows where substrate is not a reactant nor a product. somewhere in middle). 4. This stage is after the reaction has occurred. Enzyme is bound to the products. 5. Enzyme is now separated from our products. Note: Binding is strongest at the transition state.

How does phosphorylation affect the function of the Na/K pump?

3 Na+ ions attache to the receptor space in the cytoplasm and once all sites are full, an ATP molecule will be broken into ADP and Pi. The free Pi will attach itself to the Na+/K+ pump and this causes the pump to change its conformation by 180 degrees to face outside the cell. The protein releases the Na+ ions into the extracellular space. Now, 2 K+ ions will attach themselves to the protein. Once all K+ sites are full another conformational change occurs. The protein will be dephosphorylated causing the protein to change in 180 degrees facing the inside of the cell. K+ ions are released into the intracellular space. Phosphorylation/Dephosphorylation regulates the activity.

What is collagen?

A structural protein with a trihelical fiber (3 left handed helices together forming a right handed helix). - makes up most of extracellular matrix of connective tissue. - provides strength and flexibility

What kind of reactions is ATP synthesis an example of?

ATP synthesis is an example of an endergonic reaction and non-spontaneous. Has a positive DG.

What is acetylation?

Acetylation is a co-translational modification where the first amino acid in a polypeptide will be removed (usually Methionine)and in its place will now be an acetyl group. A "small" post-translational modification.

Enzymes speed up reactions. How is a reaction altered by the catalytic strategy of acid/base catalysis?

Acids and bases are proton donors or acceptors. Ex. Keto-enol tautomerization - a proton moving from a carbon atom to an oxygen atom. Acids and bases are good proton carriers and donors they can help the reaction go quickly by moving the proton around instead of the molecule doing it by itself.

How do active sites bind substrates?

Active sites usually bind substrates reversibly via a non-covalent bond. These non-covalent electric forces (hydrogen bonds, van der Waals, hydrophobic effect) can promote the reversible binding between active site and the substrates.

What is an enzyme and its purpose for dissolving carbon dioxide into the blood?

An enzyme is a biological molecule that acts to catalyze the wide range of reactions that take place in our cells. Life would be unstable if enzymes did not exist. Enzymes greatly increase the rate of a reaction. - Alters the rxn path only, not the reactants or products. [ Ex. Carbonic anhydrase speeds up the conversion of carbon dioxide and water into carbonic acid that dissociates into hydrogen ions and polar bicarbonate ions. Without this enzyme, carbon dioxide could not be dissolved into the blood for transport. ]

What are antibodies and describe their structure?

Antibodies (Immunoglobins) are protein components of the adaptive immune system which binds to antigens and targets them for destruction. An antibody has a strong affinity for its ligand. Antigen is the antibodies ligand. They are Y shaped proteins made of 2 identical heavy chains and 2 identical light chains.

Which of the following are true about enzymes? I. Enzymes bind and inhibit the cooperativity between substrates and active sites. II. Enzymes bind to specific reactants (substrates) and catalyze a single reaction or a set of related reactions. III. Enzymes speed up chemical reactions without lowering the activation energy. IV. Enzymes are highly efficient and limit the number of unwanted products. A) I and IV B) II and IV C) III only D) I , II, IV

B) II and IV

What cell produces immunoglobins?

B-cells and they function to neutralize targets in the body and recruit other cells to eliminate a threat.

What are cell adhesion molecules (CAMs)?

CAMs are proteins on the cell surface of most cells and aid in binding the cell to the extracellular matrix. CAMs are all integral membrane proteins. 3 categories of CAMs: - cadherins - integrins - selectins

What is the major difference between co-factors and co-enzymes?

Co-enzymes - carrier molecules Co-factors - assist with catalysis

What are Co-translational modifications?

Co-translational modifications are occurring to the growing polypeptide chain as it is being translated.

What kind of reactions are combustion reactions an example of?

Combustion reactions are examples of exergonic and spontaneous. Has a negative DG.

Enzymes require specific environments for them to work properly. What would happen to DNA Pol at normal pH of 7 vs. if the pH was reduced to about 2?

DNA is a negatively charged molecule. DNA Pol uses Mg2+ as a cofactor for it to replicate the DNA. Normal pH conditions, the DNA Pol will hold on to the Mg2+ ion through electrostatic interactions between Mg2+ and one of its aspartate residues, thus deprotonated the DNA Pol Aspartate residue and negatively charged at neutral pH values. Now, put DNA Pol into a reduced pH environment. The Asp residue will become protonated. Asp now doesn't have a negative charge and it can no longer perform its job. Loss of functionality!

What interaction holds the heavy and light chains together in an antibody?

Disulfide linkages and noncovalent interactions.

Enzymes do not typically function alone so what is required for them to function?

Enzymes require helper molecules. Cofactors are helper molecules that are needed for the enzyme to properly function. Cofactors are organic carrier molecules that are primarily carbon molecules. Cofactors are different than co-enzymes since they are directly involved in the catalysis and stabilizing the reactants. Ex. NADH = NAD+ + H:- (carries electrons)

True/False? All proteins are classified as an enzyme or a non-enzyme protein.

False, Not all enzymes are able to be classified into one of the two categories. Sometimes a protein can have properties of both enzymes and non-enzyme proteins.

True/False? Enzymes are not specific to the reaction type, they just speed up the process.

False, Enzymes are highly specific.

True/False? In a catalyzed reaction, the activation energy is lowered which means the amount of free energy is also lowered.

False, Gibbs free energy depends on the energy of the products and reactants. It is independent of the pathway taken to convert the reactants to products.

True/False? All enzymes are proteins.

False, Most enzymes are polypeptides, however RNA molecules can also act as catalysts.

True/False? The catalytic enzyme in a reaction has no affect on the stability of the reaction.

False, The catalyzed reaction has a transition state with a much lower energy, so it is more stable with the enzyme The reaction as a whole has a lower Free Energy of Activation.

True/False? Phosphorylation can occur anywhere as long as there is ATP and a kinase.

False, Phosphorylation of proteins does not occur outside the cell in the extracellular space because there is not an abundance of ATP. Phosphorylation occurs inside cells where the concentration of ATP is abundant.

True/False? Enzymes can have other sites called allosteric sites, that do not play a role in its funciton.

False, The enzyme can have other important sites called allosteric sites that may play regulatory roles.

True/False? Enzymes are named for their reactions.

False; Enzymes are sometimes named for their reactions. They are not always named this way.

Describe the difference between Gibbs Free Energy and the Activation Energy (Freen Energy of Activation) of a reaction.

Gibbs Free Energy is the difference in energy between the reactants and products. - talks about where equilibrium will be achieved. - Activation Energy describes how quickly a reaction take place. - talks about how quickly the equilibrium will be achieved - DOES NOT alter the equilibrium of a reaction (DG) - also describes the kinetics (speed, rate) of the reaction.

What is the result of glycosylation on a protein?

Glycosylation is adding a carbohydrate to a protein. Glycosylation usually occurs to proteins that are embedded in the membrane. Glycosylation can be used to identify different types of cells. Ex. ABO blood groups. A "small" post-translational modification.

What is a haloenzyme?

Haloenzymes are enzymes bound to a cofactor.

Enzymes speed up reactions. How is a reaction altered by the catalytic strategy of covalent catalysis?

Happens when enzymes form a covalent bond with another molecule usually their target molecule. A decarboxylation reaction (a CO2 group is taken off a molecule) has a lot of electrons moving around. A covalently bound enzyme that could hold on to some electrons like an electron carrier or electron sink could then help the reaction occur more quickly.

What kind of reaction does a hydrolase catalyze?

Hydrolase is a type of enzyme that uses water molecules to cleave one molecule like A into two other molecules such as B and C. An example of a good hydrolase is the hydrolysis reaction that can occur between peptide bonds. Lys-Ala + H2O yields Lys + Ala - serine hydrolases/("protease") these are hydrolases that use a serine residue as the key catalytic amino acid for breaking peptide bonds.

Which of the following about enzymes is/are true? I. Enzymes can bind more than one substrate like pyruvate and NADH. II. Allosteric binding is just the binding of an inhibiting substrate molecule to the active site. III. An inhibitor (regulating molecule) can bind to an enzyme at a different location than its active site. IV. An allosteric binding site is just any site where a regulating molecule can bind that is not part of the active site. V. Enzymes can bind regulators at their allosteric sites which can change the structure/shape of the active site. VI. Regulating molecules bind when the enzyme needs to stop its function.

I , III, IV, V I. Enzymes can bind more than one substrate like pyruvate and NADH. III. An inhibitor (regulating molecule) can bind to an enzyme at a different location than its active site. IV. An allosteric binding site is just any site where a regulating molecule can bind that is not part of the active site. V. Enzymes can bind regulators at their allosteric sites which can change the structure/shape of the active site. NOTE: Allosteric binding is when a regulatory molecule (an inhibitor or activator) binds to an enzyme at a site that is not its active site.

What is the energy in glucose made from plants used for in animals?

In animals, the energy stored in the chemical bonds of glucose are transformed into the energy stored in the proton gradient across the mitochondrial membrane and this energy is then transformed into chemical bonds of ATP.

Why do active sites need to have a complementary structure similar to their substrates?

In order for the meaningful non-covalent interactions between the residues of the active site and the substrate to be meaningful and strong enough, the distances between them must be short enough. So, the substrate must fit snuggly into the active site. 2 models explain this binding: Lock and key model and the induced fit model.

Enzymes speed up reactions. How is a reaction altered by the catalytic strategy of proximity and orientation effects?

In order for two reactants or subunits to react with each other, they need to physically collide at some point. The orientation of the two colliding molecules must be in the correct position for the collision to result in a successful reaction. This helps increase the frequency of collision.

How do enzymes transform radiation into usable chemical energy in plants?

In photosynthesis, plants use a variety of enzymes that capture the electromagnetic radiation from the sun and transform it into a more usable form that is stored in chemical bonds like in glucose. Humans and other animals eat the glucose and gain the energy from the chemical bonds

What is the lock and key model and how does it relate to an enzyme's active sites?

In this model the substrate fits precisely and perfectly into the active site due to their exact complementary shapes. Ex. A lock and a key.

What kind of reaction does an isomerase catalyze?

Isomerase is an enzyme group that typically converts a reactant into one of its isomers. An example of this is the conversion of Glucose-6-P into Fructose-6-P by Phosphoglucose isomerase

What is an enzyme called if it is not bound to its cofactor?

It is an Apoenzyme.

What is the Standard Free Energy Change of a reaction?

It represents the net change in energy levels between the reactants and products It is also the energy that is released into the environment when the reaction is over. Products at a lower energy state than the reactants means the reaction is spontaneous. - Can be written as Erxn

What are the motor proteins that are associated with microtubules and what are their functions?

Kinesin and dynein both: - have 2 heads - one attached to tubulin at all times. - important for vesicle transport - have opposite polarities: kinesins bring vesicles to positive end of cell and dyneins bring them to the negative side. Kinesin - role in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis. Dyneins - sliding movement of cilia and flagella - retrograde transport.

What kind of reaction does a Ligase catalyze?

Ligases catalyze reactions where two molecules are combining to form a complex between the two. An example of this reaction occurs during DNA replication where 2 strands of DNA are joined together. (DNA Ligase).

What is lipidation?

Lipidation is adding a lipid to a protein that will be attached to the cell membrane. GPI anchors are lipids that help to attach proteins to the cell membrane. This is important because the protein has both polar and non-polar parts. The GPI anchor plunges into the hydrophobic part of the cell membrane. Usually occurs in the Endoplasmic reticulum and Golgi Apparatus, but not all.

What kind of reaction does a Lyase catalyze?

Lyase is an enzyme group that catalyzes the dissociation of a molecule into multiple products without using water like hydrolases and also without using oxidation and reduction like an oxidoreductase so they need to generate a double bond or a ring structure between two atoms to work. An example of a lyase is the cleavage of Argininosuccinate into Arginine + Succinate by Argininosuccinate Lyase

What would be the effects of Lysine acetylation?

Lysine has an extra amino acid on its side group that can act as a base and carries a positive charge. If Lysine were to be acetylated (a covalent modification) on the amino and nitrogen, the electron withdrawing effect of the acetyl group will prevent the nitrogen from carrying a positive charge, modifying the behavior of the amino acid. The loss of the amino acids charge can change a few properties: acidity/basicity; electrostatic interactions;

Which protein modification is typically found on histones and associated with gene regulation?

Methylation of histones. DNA wraps around histones for packaging. Methylating and de-methylating histones can turn certain genes on and off, regulating the activity of genes here instead of proteins. A "small" post-translational modification.

Suppose you have a reaction beaker that is filled with 100 enzyme molecules. What would be the predicted enzyme activity of the reaction if you add 50 substrate molecules? What would be the predicted enzyme activity of the reaction if you add 100 substrate molecules?

Only 50 enzymes will have filled active sites. The entire activity of all the enzymes will be half of its maximum value. Now adding 100 substrate molecules, then the entire activity of all the enzymes will be filled indicating a maximum enzyme velocity.

What kind of reaction does an oxidoreductase catalyze?

Oxidoreductase is unique compared to the other types of enzymes because it involves 2 types of reactions. They catalyze reactions where electrons from molecule B are moved to molecule A or electrons from molecule A are transferred to molecule B. Usually can catalyze forward and reverse reactions. An oxidase is directly involved in oxidizing or removing electrons from a molecule, whereas a reductase is involved in reducing or adding electrons to a molecule. (lactic acid fermentation = lactate dehydrogenase)

What translational modification is the preferred method of protein modification?

Phosphorylation is a very effective, convenient and an efficient process.

What is phosphorylation?

Phosphorylation is the most common type of post-translational modification. Phosphorylation is the adding of a phosphate group and dephosphorylation is the removal of a phosphate group. Also called a covalent modification.

What are post-translational modifications?

Post-translational (covalent) modifications contains the majority of all protein modifications. These modifications happen after the polypeptide is translated. Some modifications occur in the Endoplasmic reticulum and Golgi Apparatus, but not all.

What enzymes are responsible for regulating and catalyzing phosphorylation and contain 100+ different homologous proteins in the human body?

Protein kinases - they phosphorylate a protein by transferring the terminal phosphoryl group from ATP onto a hydroxyl containing residue.

What is proteolysis and its function?

Proteolysis is a protein modification that is sometimes cut before it can be functional. A zymogen is an inactive form of a protein and the only way to activate it is by cutting it.

Enzymes speed up reactions. How is a reaction altered by the catalytic strategy of electrostatic catalysis?

Recall DNA is a negatively charged polymer because of the negatively charge phosphates in the backbone. An enzyme that has a metal cation like magnesium could help stabilize the negative charge in DNA to help make it easier to work with. DNA polymerase is an example.

What is the difference between a receptor and an ion channel?

Receptors are proteins that receive or bind a signaling molecule like a ligand for ex. insulin and span the entire membrane bilayer. Ion Channels span then entire membrane bilayer. This protein acts as a pore or channel for ions to enter or exit the cell.

The constant region of the antibody has the ability to:

Recruit and bind other cells of the immune system like macrophages.

What is reversible binding?

Reversible binding is when the substrate binds to the active site and the substrate is changed into the product it will detach itself moving away from the active site. It does not remain bound to the active site forever.

Which amino acids are able to be phosphorylated?

Serine, Threonine, Tyrosine

What is the Free Energy of Activation?

The activation energy is the difference between the energy level of where we start and the top of our graph at the transition state. The Free Energy of Activation is the amount of energy A needs to have to break the barrier to get to point B This energy value typically indicates how quickly a reaction will occur. Can also be written as EsubA.

What is the active site on an enzyme?

The active site is a specific region on an enzyme that binds to the substrate. It is a 3D crack in the enzyme that contains catalytic groups - the amino acid residues that come from different parts of the enzyme and which catalyze the actual reaction.

Why is the active site of an enzyme smaller than the overall enzyme?

The active site is much smaller than the actual size of the enzyme. The remaining portion of the enzyme acts to create and support the active site by bringing residues that are far apart close together. The entire enzyme creates a scaffolding system that supports and stabilizes the small active site to catalyze the reaction.

How do active sites stabilize the transition state?

The active sites on an enzyme contains the catalytic residues responsible for lowering the free energy of transition state and stimulates bond breaking/forming.

What is the Transition State of a reaction?

The highest energy point on the path from A to B. It is where you will find the most instability of the entire reaction. It does not exist for a long time and is unstable. It is represented by the double dagger symbol.

What is the induced fit model and how does it relate to an enzyme's active sites?

The induced fit model describes more correctly the way the binding takes place. In this model, the shape of the enzyme's active sites is not exactly complementary, but upon binding of a substrate to the active site, the binding causes the active site to become complementary to the substrate. Ex. Changes or induces shape of active site to fit substrate.

What are the primary structural proteins in the body and what is a structural protein?

The primary structural proteins in the body are collagen, elastin, keratin, actin and tubulin. Structural proteins generally have high repetative secondary structures known as a motif, giving a fibrous nature.

How does an enzyme affect the kinetics of a reaction indicating how an uncatalyzed reaction is changed to a catalyzed reaction.

The speed of the reaction is determined by the activation energy. In an uncatalyzed reaction, there is a partially broken bond and a partially formed bond between the reactants in an uncatalyzed reaction. The electron densities of the reactants are not overlapping very well, which increases the energy of the transition state. In a catalyzed reaction, the enzyme takes in the reactants that bind to its active site. The enzyme then stabilizes the partially formed and broken bonds of the reactants. The stabilization of the transition state stimulates the breakage of old bonds and formation of new bonds. This results in a lowering of its activation energy to increase the reaction rate.

What are the 2 categories of cofactors?

The two categories of cofactors are metal ions and organic molecules called coenzymes - usually formed from vitamins.

Why is the antigen-binding region significant on an antibody?

These regions are the tips of the "Y". They are specific polypeptide sequences that will bind only one specific antigen sequence. The remaining part of the Ig is the constant region.

What are zymogens?

They are inactive enzymes that require covalent modification to become active. Ex. Digestive enzymes like trypsinogen released by the pancreas. Trypsinogen -[Enterokinase] - Trypsin

What kind of reaction does a transferase catalyze?

They catalyze reactions that move some functional group X from molecule A to molecule B. Most commonly found in protein translation, where amino acids from tRNA are transferred to the growing peptide chain. (peptidyltransferase)

What is the function of transport proteins?

Transport proteins are responsible for binding small molecules and transporting them to other locations in multicellular organisms. These proteins must have a high affinity for their ligand when the ligand is in high concentrations. At low concentration of ligand the transport protein has a low affinity for the ligand. Ex. Hemoglobin picks up oxygen in the lungs and delivers it to tissues.

True/False? A coenzyme can bind to other enzymes weakly or strongly. The tightly bound coenzymes are called prosthetic groups.

True

True/False? Enzymes typically transform one energy form into a more usable form.

True

True/False? The body is constantly undergoing biochemical reactions and enzymes function to help the reaction go by faster.

True

Enzymes are not used up in a reaction but their shape can be changed at the end of the reaction.

True, Enzymes are never used up in a reaction. The shape of the enzyme can be changed in a reaction, but it will return back to its original structure.

What is the enzyme Trypsin and its function?

Trypsin is a digestive enzyme, that binds to polypeptides and carries out two closely related reactions. It catalyzes the cleavage of peptide bonds on the carboxyl side of lysine and arginine amino acids.

Which protein modification marks a protein for degradation?

Ubiquitination is a protein modification that marks a protein for degradation.

What is allosteric binding?

When a regulating molecule, like an inhibitor, binds in a different location other than an active site. This changes the shape of the enzyme so that it can no longer carry out the reaction.

What mechanisms and characteristics are involved in making the microenvironment after the substrate binds to the active site?

When the substrate binds to the active site, the enzyme slightly closes creating a non-polar microenvironment for bonds to be broken and formed more easily. Water is only found in the microenvironment when it is a reactant in the specific reaction. The active site brings the reactants close together to form the favored product and prevent unwanted products to be formed.

What is referred to as Dietary co-factors and co-enzymes?

Yes, vitamins and minerals are not made by the body and must be ingested through the diet. Vitamins and minerals are various co-factors and co-enzymes. Vitamins = are organic co-factors and co-enzymes VitB3 shown as niacin representing NAD VitB5 for CoA Minerals are inorganic co-factors Magnesium for DNA Pol Calcium for bones and teeth structure.

What are the two factors that generally are concerned with an enzymes environment?

pH and Temperature.