Ls3 - Lecture 4

Front 1

What is an enzyme?

Back 1

Enzymes are proteins that catalyze reactions. These proteins act as highly specific catalysts. They contain an active site that brings substrates (ligands) together and facilitates the reaction. They increase the rate of a reaction. When the rate of a reaction is increased, equilibrium is also reached faster.

Front 2

What do enzymes need to catalyze a reaction?

Back 2

- An enzyme catalyzes a reaction by providing a specific environment in which the reaction can occur more rapidly. A molecule that undergoes an enzyme-catalyzed reaction is referred to as a substrate. A substrate differs from a ligand in that it can undergo a chemical transformation while bound to the enzyme, whereas a ligand does not.

-The substrate interacts with the enzyme in a pocket known as the active site. The active site is typically lined with multiple chemical groups—amino acid side chains, metal ion cofactors, and/or coenzymes—all oriented to facilitate the reaction.

- An enzyme-catalyzed reaction is highly specific for that particular reaction.

- Catalysis often requires conformational flexibility.

- Many enzymes are regulated.

Front 3

Most of the energy required to increase reaction rates by enzymes comes from what?

Back 3

Much of the energy required to increase the reaction rate is derived from weak, noncovalent interactions between substrate and enzyme, including hydrogen bonds and hydrophobic and ionic interactions. The formation of each weak interaction is accompanied by the release of a small amount of free energy that stabilizes the interaction. The energy derived from enzyme-substrate interaction is called binding energy, ΔGB.

Front 4

Homeostasis among enzymes

Back 4

There is a homeostasis between substrates & the products. The enzyme can function over again and not be changed by the reaction.

Front 5

How do products differ from the substrates?

Back 5

A product differs from substrates by one or more covalent bonds.

Front 6

How many substrates can an enzyme bind?

Back 6

It can only bind as much active sites it has. Increasing the amount of substrates won't speed up a reaction forever. It will only speed up as long as there are enough active sites, it will plateau eventually

Front 7

What does the name of an enzyme tell you?

Back 7

The name of an enzyme indicates its substrates/function (Ribonuclease, protease, kinase etc).

Front 8

What can you describe from this picture?

Back 8

.

Front 9

What is the activation energy?

Back 9

The difference between the energy levels of the ground state and the transition state. The energy required to get over the hill and to start the reaction. It determines the speed of the reaction. The rate of a reaction depends on the height of the energy hill that separates the product from the substrate. A higher activation energy corresponds to a slower reaction.

Front 10

What is Delta G?

Back 10

The difference between the substrate's start and where the product is. Delta G only determines the direction of a reaction, NOT the rate.

Front 11

Exergonic Reaction

Back 11

Delta G is negative. This doesn't need energy to go forward. Will do so without energy. Also known as a spontaneous reaction.

Front 12

Endergonic Reaction

Back 12

Delta G is positive. Needs energy to start.

Front 13

How do catalysts lower the activation energy?

Back 13

They interact with the transition state. The substrates must be in a certain orientation. The enzyme has active sites that bind the substrates, and the enzyme will undergo a conformational change due to the strain of having substrates at the active site. This moves the substrates into locations that are more energetically favorable to undergo reactions. Bonds will change and once substrates are released, the changed substrates are now products. R groups stabilize enzymes.

Front 14

How do enzymes stabilize transition states and catalyze reactions?

Back 14

By facilitating: Orienting the substrates correctly to each other, changing in reactivity of substrates to and inducing strain on substrates.

Front 15

How do enzymes use non-covalent bonding to increase reaction rates?

Back 15

Much of the catalytic power of an enzyme is ultimately derived from the free energy released in forming many weak bonds and interactions between the enzyme and its substrate. This binding energy contributes to specificity as well as to catalysis. Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to substrates per se but to the transition states through which substrates pass as they are converted to products during the reaction. The weak interactions formed only in the transition state are those that make the primary contribution to catalysis

Front 16

Explain the breakdown of alcohol and the cause of alcohol flush

Back 16

Ethanol (alcohol) is broken down in two steps: It's first converted to acetaldehyde then acetic acid. Within this, there are two versions of the enzyme that convert acetaldehyde into acetic acid. One is a fast reaction and the other is a slow reaction. The slow reaction is the one that results in the alcohol flush and it's genetic. This is due to a single amino acid difference. Because one works slow, there is a buildup of acetylaldehyde that causes the flush.

Front 17

What happens to the reactions that are energetically unfavorable?

Back 17

Enzymes couple endergonic reactions with exergonic ones. This makes overall reactions exergonic (spontaneous).

Front 18

What is the most common exergonic reaction that is coupled with the endergonic reaction to convert it into an exergonic reaction?

Back 18

ATP Hydrolysis

Front 19

How does ATP hydrolysis work?

Back 19

In an uncoupled reaction ATP hydrolysis, energy is lost as heat. If combined with another reaction, it wont be lost as heat, and this energy can be used for something else.

A phosphate is transferred from ATP to one of the substrates - creating an active substrate. The bond to the phosphate group contains high energy. This energy can then be used to facilitate the endergonic reaction of A + B.

The creation/breaking of phosphodiester bonds by water releases energy for coupled reactions.

Front 20

Where and how do coupled reactions occur?

Back 20

Substrates and ATP will combine. One phosphate group will be removed and added to substrate. Then we lose the phosphate to make reaction more forward.

Front 21

What are some limits to catalysis?

Back 21

There is a max substrate concentration that a set concentration of enzymes can handle. This is different from uncatalyzed reactions because increasing the substrate once would increase the product.

Front 22

How are enzymes regulated?

Back 22

Feedback inhibition, allosteric interactions, competitive inhibition, enzyme modifications and enzyme levels.

Front 23

Feedback Inhibition

Back 23

There is a metabolic pathway. An enzyme binds substances and makes end products. The product can bind to the enzyme in a different site. The enzyme has active sites & feedback sites. If too much product is made, it will bind to feedback site and undergo conformational change to conserve energy & stop products from being made.

Front 24

Allosteric Regulation

Back 24

A separate protein, called regulatory molecule, can bind to an enzyme and turn it's functions on/off. It will bind to a location other than the active site & change the active sites shape. The active site can now bind substrates & perform the reaction. This can lead to either inhibition or activation of an enzyme.

Front 25

Competitive Inhibition

Back 25

Competitive inhibitor (regulatory molecule) blocks the substrate from binding to an enzyme. It competes with the substrate for the active site. It prevents the substrate and enzyme from binding. It is structurally similar to the original substrate and mimics the substrate to be able to control the enzyme. This will either slow the reaction or stop it. It's also a type of reversible inhibition.

Front 26

Enzyme regulation via protein modification

Back 26

Otherwise known as covalent modification of one or more amino acid residues in a protein molecule. To regulate enzymes, you can add a phosphate (most common) for the protein to get phosphorylated by hydrolysis. Phosphate will be added to the R group of the protein, or the reverse can be done. This can activate or inactivate the protein, respectively. Other possible forms are adenylation, acetylation etc. DNA & protein interact with each other as well to regulate enzymes. These varied groups are generally linked to and removed from a regulated enzyme or other protein by separate enzymes. When an amino acid residue is modified, a novel amino acid with altered properties is effectively introduced into the protein.The R group can bind with base pairs of the DNA.

Front 27

Protein modification - Phosphate

Back 27

The attachment of phosphoryl groups to specific amino acid residues of a protein is catalyzed by protein kinases; the removal of the groups is catalyzed by protein phosphatases. The oxygen atoms of a phosphoryl group can hydrogen-bond with one or several groups in a protein, commonly the amide groups of the peptide backbone at the start of an a helix or the charged guanidinium group of an Arg residue. To serve as an effective regulatory mechanism, phosphorylation must be reversible.

Front 28

*What are non-specific DNA-Binding proteins?

Back 28

Bind to DNA nonspecifically, independent of DNA sequence. “Nonspecific” DNA-binding proteins often display a measurable bias for binding of DNA sequences with particular features. Nonspecific DNA-binding proteins exhibit only limited hydrogen-bonding interactions with bases in the DNA. Instead, electrostatic interactions with the negatively charged phosphate groups, hydrogen bonds to the backbone deoxyribose, and the nonspecific hydrophobic effect with the bases predominate, to varying degrees

Front 29

*What are specific DNA-Binding proteins?

Back 29

Recognize particular DNA sequences and bind tightly at the genomic locations where those sequences occur. Proteins that bind with an enhanced affinity to particular DNA sequences are critical to the regulation of many processes in DNA metabolism.To bind to specific DNA sequences, regulatory proteins must recognize and distinguish surface features on the DNA. Most of the chemical groups that differ among the four bases and thus permit discrimination between base pairs are hydrogen-bond donor and acceptor groups exposed in the major groove of DNA. Most of the protein-DNA contacts that impart specificity are hydrogen bonds.Protein-DNA contacts are also possible in the minor groove of DNA, but the hydrogen-bonding patterns there generally do not allow ready discrimination between base pairs.

Front 30

*Specific DNA-Binding Protein: Lac Repressor

Back 30

When the Lac repressor is not bound to the operon’s promoter region, RNA polymerase binds to the promoter and transcribes several linked genes, here labeled A, B, and C. The Lac repressor binds to two operators on either side of the promoter to shut down transcription, apparently forming a loop in the DNA that prevents RNA polymerase from binding to the promoter. Each operator consists of an inverted repeat (top inset). The helix-turn-helix motif of the repressor protein binds specifically in the major groove of the operator recognition sequences (bottom inset). The repressor discriminates between the operators and other sequences, so binding to these few dozen base pairs among the 4.6 million or so of the E. coli chromosome is highly specific.

Front 31

Enzyme Levels

Back 31

The level of activity of a certain enzyme can be affected by both the rate of enzyme synthesis and the rate of enzyme breakdown. Transcription and translation can be very important in determining the regulation of enzymes. The breakdown of proteins is also important for determining the concentration of enzymes. Enzymes could also just not be made.

Front 32

How can we analyze proteins?

Back 32

We analyze proteins to see if the protein is in a cell/not in the cell etc. There are different methods to cell lysis to release proteins from the cell. We want to isolate proteins to be able to analyze them.

Front 33

How can we break cells and tissues & create a homogenate to analyze them?

Back 33

To make a homogenate, the first step in the purification of most proteins is to disrupt tissues and cells in a controlled fashion. Using gentle mechanical procedures, called homogenization, the plasma membrane of cells can be ruptured so that the cell contents are released. The following are 4 common procedures:

- Break cells with high frequency sound

- Use a mild detergent to make holes in the plasma membrane

- Force cells through a small hole using high pressure

- Shear cells between close-fitting rotating plunger and the thick walls of a glass vessel.

Front 34

What is centrifugation?

Back 34

Centrifugation separates particles by mass and shape/density. Anything with a high density/mass will be at the bottom of the tube. Anything in cytoplasm will be in the supernatent (this includes the proteins because they aren't heavy).

Front 35

What is differential centrifugation?

Back 35

In differential centrifugation, we can use only the supernatent and spin at different rates to target specific organelles.

Basically centrifuge at different rates to isolate smaller and smaller cellular materials. The pellet settles to the bottom and they take the supernatant and centrifuge that at a higher rate. That creates another pellet and supernatant. They then centrifuge the supernatant again

Front 36

What is density gradient centrifugation?

Back 36

This will actually isolate specific organelles within the tube.

Create a density gradient of a substance like sucrose where it’s less dense on top and more dense on the bottom. The homogenate is added and then centrifuged. The parts of the cell will sink to the equivalent sucrose density, separating them by size.

Front 37

What happens after fractionation?

Back 37

Fractionating will separate proteins by properties. You will put proteins in columns with a solvent with certain properties. This will separate proteins based on properties. There are 3 ways to do this: Ion-Exchange Chromatography, Gel Filtration Chromatography, Affinity Chromatography

Front 38

What is chromatography?

Back 38

There are many ways to separate proteins. One method is chromatography, and one of the most powerful chromatographic techniques is column chromatography. In this technique, a protein mixture is applied to a column containing a resin, or matrix,that interacts differently with the various proteins. After the protein solution is applied to the top of the column, a buffer is passed through the column to thoroughly wash away any proteins that do not bind to the matrix. Then another buffer is applied that causes bound proteins to dissociate from the matrix; the proteins are carried out in the buffer flow, a process referred to as “elution” of proteins from the column. The proteins come off the column at different times, depending on how they interact with the resin. The column matrix and “elution buffer” are carefully chosen so that different proteins dissociate from the matrix at different times. The eluted proteins are collected in a fraction collector, which gradually moves test tubes under the column, thus keeping the proteins that elute at different times separate from one another.

Front 39

Ion-Exchange Chromatography

Back 39

This separates proteins by charge. Their is a column with beads in it. The beads have either a (-) or (+) charge. Depending on the charge of the beams, we can separate proteins. In this case, lets choose (-) beads. (+) proteins will bond to the beads, but not (-) proteins. First things that drop out of column are most (-) charged proteins, left with just (+) charged proteins in the end.

Front 40

Gel Filtration Chromatography

Back 40

This separates proteins by size. The beads have pores with different sizes. Small proteins will get stuck in pores and large proteins will leave the tube first. Medium ones will come next cuz they do bind a little bit with pores.

Front 41

Affinity Chromatography

Back 41

Used to purify proteins. Uses antibodies (very specific for protein of interest). Antibody will attach to bead. Protein recognized by the antibody. All other proteins not recognized by antibody will go towards the bottom.