BB 450 Unit 2

What is activation energy? How does it change the energy need for rxn and what does not change?

Activation Energy: The energy necessary for an uncatalyzed reaction to occur. The minimum quantity of energy which the reacting species must possess in order to undergo a specified reaction. Using an enzyme will lower the activation energy. The *overall* energy of the reaction doesn't change it's just that the enzyme lowered the energy required to initiate the reaction.

Describe allosteric regulation of enzymes? Is it positive or negative?

Allosterism: binding of a small molecule to an enzyme that affects the enzyme activity (it's affinity to the substrate) Homotrophic Effector: A substrate for the enzyme Heterotrophic Effector: A non-substrate molecule Regulation could be either positive (more binding) or negative (less binding)

How are the catalytic triads of other types of proteases similar to and different from serine proteases?

Aspartyl: * Uses 2 Aspartic Acids * Has a substrate binding site similar to the S1 pocket * One of the carboxyl groups on the aspartic acid become ionized and the other does not, so there's an electronic environment that dictates this. There's not much water in here so water doesn't dictate the behavior. * Aspartic Acid grabs a proton off of water when it can and that's how this reaction happens Cysteine: * Similar to Serine Proteases but they use just 2 AA * Histidine and Cysteine Metalloproteases: * Electron Rich side chain that helps activate water * Metal that helps hold onto water while it's being activated

How are restriction enzymes used in the lab? What do they cut? Where do they cut?

Can chop up DNA and you can look if you have the DNA you thought you did by chopping with specific endonucleases. Restriction Enzymes cut DNA The cuts are always made at specific nucleotide sequences. Different restriction enzymes recognize and cut different DNA sequences. The cut occurs where the DNA bends. Water is activated and there's a nucleophilic attack. The intermediate falls apart and then more H2O comes in. Eventually both strands of DNA are broken/cleaved.

What is the role of carbonic anhydrase in protecting us from CO2?

Carbonic anhydrase dissolve CO2 in the blood Recall some CO2 gets moved into our lungs via binding to hemoglobin but that's not the only way this occurs. The major way is that the bicarbonate ion is dissolved in the blood. CO2 is poisonous to us, and so dissolving it/getting rid of it is important The Kcat differs depending on pH. Blood should have a certain pH but these molecules being dissolved in it can have an effect on that.

Note how they talk about a catalytic dyad.

Cis-145, holds the ligand really tight but it is reversible His41 and Cys145 is important for the proteolytic activity of SARS-CoV-2 3CLpro

What is an enzyme cofactor and what are the types?

Cofactors help to catalyze a reaction faster or make them happen at all The types are coenzymes and metals

Contrast competitive with noncompetitive inhibition

Competitive Inhibitors cross the original Lineweaver-Burk plot at the y axis (competitive inhibitors cross competitively) Competitive has the original Vmax while non competitive has the original Km

Enzyme inhibition - what are the types?

Competitive and Noncompetitive

What does the line look like with various inhibitors?

Competitive: Steeper slope, same Vmax, Larger Km Non-Competitive: Steeper slope, decreased Vmax, same Km

Know some protease families and what types of proteases belong?

Cysteine: Papain, Cathepsin K, Calpain, Caspase. Important for cellular signaling. They can chop things up and are very specific in what they chop. Caspases in some cases will initiate apoptosis Aspartyl: Cathepsin D, Cathepsin E, Pepsin Pepsin is in the stomach and works at a pH of about 2 Metallo- : Carboxypeptidase A (chews away at the carboxy terminus rather than cleaving in the middle), Collagenases (act in digestion bc. we eat things that have collagen)

What is a random reaction?

Either substrate can bind first, there is no binding order. (Multiple Substrates)

Is the enzyme changed by the reaction?

Enzymes are *not* permanently changed by the reaction but while it is happening there is some conformational changes. But once it releases the substrate it goes back.

What is a perfect enzyme? Examples?

Enzymes that work really rapidly that they're only limitations are due to diffusion Ex. Acetylcholinesterase, Carbonic Anhydrase, Catalase, Crotonase, Fumerase, Triose phosphate isomerase, Beta-lactamase, Superoxide dismutase

What is the reason we need metals in our diet?

Essential metals activate enzymes that are required to perform some bodily functions. Often metals are used in tiny amounts because they can be toxic in our body

Why is it given IV rather than orally/digestive tract?

For better GI Tract absorption

How is the solubility of this drug improved?

Having the phosphate croup improves solubility because it increases the total polar surface area and lowers the log d or log p

What does tobacco smoking do to alpha-1-antitrypsin? What is the physiological result?

If a person is a smoker they create a lot of reactive oxygen species which produces a hydroxyl radical which attacks the sulfur of methionine and creates a sulfoxide. If alpha-1-antitrypsin ends up with it's methionine oxidized then it can't interact with elastase. If elastase is loose it can degrade the lung.

How does noncompetitive inhibition work? What happens to Vmax and Km?

Inhibitors don't resemble the substrate and they don't bind to the active site. They bind to another site on the enzyme. This binding yields a shape change that makes the substrate unable to bind to the enzyme bc. the enzyme now has a weird pocket that the substrate can't recognize. The inhibitor can't be out competed by adding more substrate bc. it can still lock up the other site The Vmax decreases Km is unaffected

What is equilibrium of a reaction? What is it not?

It IS the point at which the concentration of the reactants and the products won't change It is NOT the point at which the concentrations of the reactants and the products won't change Unless delta G naught prime = 0 than it is WRONG to say [A] at t = 0 = [B] at t = 0

Why did TPI evolve to be a perfect enzyme?

It catalyzes a reaction from glycolysis The intermediate is unstable and it can fall apart and if it does fall apart it becomes a poison and toxic. this enzyme actually evolved to work really rapidly to prevent this consequence and in turn that is how it became a perfect enzyme

What is initial velocity? How does velocity change?

It is measured as [Product] / Time It changes depending on the [S] (molarity)

What is a Lineweaver-Burk plot used for? What does it plot? Why would you want to use one?

It is used to visualize Michaelis Menten Kinetics It is a double reciprocal plot that plots 1/V0 and 1/[S] You would want to use it to get direct readings of -1/Km (x-int) and 1/Vmax (y-int)

What is ping pong or double displacement reaction? Can you recognize it?

It is when one group on each of the substrates swap places with each other. The enzyme flips between the two states with the two different attached groups.

What are the enzyme kinetics for a competitive inhibitor? What happens to Vmax and Km?

It lowers the Velocity buildup, it will eventually reach the same Vmax but at low amounts of substrate the gap btw the inhibited and non inhibited lines are larger but then the more substrate the smaller that gap gets. Because w/ more substrate there's a higher likelihood that the enzyme will bind to a substrate rather than an inhibitor. Vmax and 1/2 V max don't change Km: it causes an apparent change in Km but binding affinity isn't affected it's just that there's less enzyme so there's an apparent change in affinity (seems to increase)

What pH does carbonic anhydrase prefer and why?

It prefers higher pH values because at higher pH it is more likely that a proton will be lost 9.0 is about the highest the enzyme can handle before it would start to become denatured

Is Km related to Vmax?

Km doesn't have a direct relationship to Vmax, Vmax is dependent upon the enzyme concentration.

How is Km related to Vmax?

Km is the concentration of substrate which permits the enzyme to achieve half Vmax.

What is the slope of a Lineweaver Burk plot?

Km/Vmax

Contrast lock and key with induced fit models? Which is true?

Lock and Key: This is the older model that posits that there's a substrate that fits into an enzyme and those shapes are an exact fit. You have to have the exact right shape of substrate or else nothing will happen. Doesn't take into account any conformational changes that may occur in the enzyme or even in a substrate. Induced Fit: Says that the enzyme changes the shape of the enzyme and vise versa (though it is a bit transient the way that the enzyme is changed). When the two components bind it creates a kind of tension/energy to it that will cause an induced fit so that they fit together even better then they did when they first came in contact The induced fit model is the more accepted one

How do enzyme kinetics differ when there are varying amounts of substrate? How does that relate to maximum velocity (Vmax)?

Low Substrate: Most of the enzyme is empty and is waiting for diffusion to give it a substrate The more substrate you have the more quickly it will find the enzyme up to a point High Substrate: When you have more substrate than some of it's going to be waiting for the enzyme to do it's job and release products. Enzyme Saturation = When there's more substrate than is needed Vmax happens at high/saturated amounts of substrate concentration

Where are the enzymes busy and where are they idle?

Low [S]: Low initial velocity, and enzymes are often idle High [S]: High initial velocity, and enzymes are almost always busy

What is an ordered reaction? What does it have to do with induced fit?

Means that something must bind first before the other one can bind. The one thing has to bind first in order to create an induced fit so that the other component can bind as well. (Multiple Substrates)

Does MM kinetics still inform us when allosterism is occurring?

No Enzymes that don't follow Michealis-Menten Kinetics include those that bind substrate cooperatively. Binding of one substrate affects the binding of others. And this happens in allosterism.

What is a protease and what does it do?

Proteases: As a category of enzyme break peptide bonds at specific sites in the amino acid sequence Enzymes that break down proteins

What are the steps of catalysis of aspartyl proteases?

1. Activation of water: The water between the two aspartate side chains are activated by pulling off a proton and creating a hydroxyl that attacks a carbonyl carbon. 2. Nucleophilic attack results in the bond breakage 3. Tetrahedral intermediate is formed that then falls apart 4. The peptide bond then breaks, there is no covalent bond here so there's no second step The aspartate side chains are projected upwards where the substrate is going to bind

How do we denote the complexes, like "ES" and "ES*" etc.?

1. E + S 2. ES 3. ES* 4. EP 5. E + P

What types of enzymes exist? What do they do?

1. Oxidoreductases: oxidation/reduction reaction catalysis. They have an electron carrier. 2. Transferases: Transfer a functional group 3. Hydrolases: Hydrolysis of bonds 4. Lyases: non-hydrolytic non-oxidative breaking of bonds 5. Isomerases: catalyze isomerization changes within a single molecule 6. Ligases: join two molecules by making covalent bonds

How does the reaction work when there are two substrates? What are the steps? (Catalysis steps)

1. The enzyme and substrates are free (E + S) 2. The substrate and the enzyme recognize and bind to each other and this forms an enzyme substrate complex (ES complex) 3. The ES complex brings the substrate closer (conformational shape change) into proximity with each other which results in a reaction between the two substrates. This is the ES* complex 4. EP Complex: The substrate changes into products 5. E + P: The products are released to go out into the cell and do jobs

What are the steps to the first half-rxn of chymotrypsin catalysis?

1. The histidine has a sink of electrons that hate being near the charges of aspartic acid so they move as far from the acid as they can get. So now they're more near Serine's proton 2. Serine is activated by the removal of a proton from the OH group, because of the e- movement from the histidine (now the Serine has a really reactive oxygen called an alkoxide ion) (Acid/Base catalysis) 3. The alkoxide nucleophile attacks the carbonyl group of the peptide bond on the substrate, cleaving the peptide chain C terminal to the phenylalanine. This causes another pocket to be formed called the oxyanion hole which stabilizes the transition state. The bond btw the carbon and nitrogen falls apart and there's a piece of that's released but the piece w/ the phenylalanine is covalently attached to the serine residue so it's still there. 4. One piece is covalently attached to the serine oxygen (acyl-enzyme intermediate) (covalent catalysis) 5. Amine leaving group is protonated and released (acid/base catalysis)

What are the steps of catalysis of cysteine proteases?

1. The thiol (SH) group on cysteine and the side chain on histidine will move closer together upon substrate binding. 2. Removal of a proton from the thiol group creating an alkoxide ion 3. The alkoxide ion searches for a nucleus and so does the sulfur group. The sulfur group is also full of electrons and is electrophilic. And the sulfur group and alkoxide ion look for the carbonyl carbon which is electron poor. 4. The peptide bond breaks and there's a tetrahedral intermediate like we saw in serine. The bond falls apart and the proton moves from the histidine's nitrogen to the amine group of the released peptide and that was the fast phase. 5. Then the second peptide needs to be removed from the sulfur, so water comes in and gets activated by the histidine and removes a proton. So the resulting hydroxl attacks the sulfur carbon bond and then another unstable intermediate is formed and the sulfur-carbon breaks and the peptide is released from that bond.

What are the steps of the second half-rxn?

1. Water binds to the active site and is activated by histidine 57 2. Water's oxygen activated by His-57 (acid/base catalysis) and as nucleophile attacks the acyl-enzyme intermediate 3. Ser195 is protonated by His57 and released (acid/base catalysis) freeing the second product and regenerating the enzyme.

How do you measure an enzyme's affinity for its substrate?

1/2 Vmax is the affinity and it takes place where the enzyme is not saturated. Km has an inverse relationship w/ enzyme affinity

How much would a chemical catalyst, as opposed to an enzyme, speed it up?

A chemical catalyst could speed things up maybe 1000 fold whereas a enzyme is closer to 1 million fold So enzymes speed up a reaction faster than an enzymatic catalyst

What is catalytic RNA?

Ribozymes: are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. Can act as enzymes

Contrast the sequential and concerted models?

Sequential: Postulates that there's a cause and effect relationship btw. binding of substrate and the enzymes changing from R to T or T to R Binding of Ligand converts 1 subunit from T to R and induces its neighbors to do the same Eventually more ligands bind and all the subunits are in the R state. Concerted: Enzyme flips naturally btw the R and T states and then when a substrate binds to it is when the structure locks into place and into the state that it prefers. It can also be stabilized by an inhibitor or an agonist which is something positive (a positive regulator)

What category of protease is chymotrypsin? Where does it cut?

Serine Protease It cuts on the C-Terminal side of aromatics phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr)

What AAs are in chymotrypsin's catalytic triad?

Serine, Histidine, and Aspartic Acid

How are serine proteases turned off?

Serpins = Serine Protease Inhibitors

In enzyme kinetics, what shape of curve results from allosterism?

Sigmoidal Allosteric Enzyme Kinetics: Substrate does change enzyme binding of substrate

What is the morpheein model?

Similar to the concerted model in the sense that they are reversible (naturally flipping btw T and R state), but instead of enzymes as a whole flipping, multisubunit enzymes first dissociate into individual subunits that reassemble and only individual units of same configuration can reassemble. Subunits are stabilized before reassembly

Examples of some types of reactions?

Single Substrate --> Single Product Single Substrate --> Multiple Products Multiple Substrate --> Single Product Multiple Substrates --> Multiple Products Then they can be ordered, random, or ping-pong

How does tension help the induced fit?

Substrate enters the active site of the enzyme and it clamps down around the substrate forming an induced fit. The tension helps because it allows for a tighter fit and more contact to occur between the enzyme and substrate Tension causes the jaws to close but the resulting change in the shape when product is created will allow the jaws to open again

What are some examples of proteases that are useful in the lab?

Subtilisin: C-terminal side of large, uncharged side chains Chymotrypsin: C-terminal side of aromatics (Phe, Tyr, Trp) Trypsin: C-terminal side of lysine and arginine that are not next to proline Carboxypeptidase: N-terminal side of C-terminal amino acid Elastase: Hydrolyzes C-terminal side of small amino acids (Gly, Ala) Cyanogen Bromide (a chemical): Hydrolyzes C-terminal side of methionine They all have they're own specificity!

Is there any type of inhibitor that binds permanently?

Suicide Inhibition: has covalent modification that sticks around for a long time. Penicillin is an example of this type of inhibition

Colorimetric assays for protease activity.

Tells us if an enzyme is active in a mixture that is made or in an extract of cells treated with a drug so you can look for the activity. The example presented is for chymotrypsin activity: There is a molecule used to see if chymotrypsin is active because it gets cut by chymotrypsin. You synthesize/purchase this molecule/peptide and you add it to your cell mixture ad if it gets cut it means that chymotrypsin is present in your original mixture and it will reflect the amount in there up to a point (enzyme kinetics). You can tell because part that gets cut turns yellow and can be seen with a spectrophotometer or plate reader. The yellow color reflects the degree of cutting that happened. There's assays for every enzyme that release every color.

How are proteases used against SARS-CoV-1 and SARS-CoV-2?

The SARS-CoV-1 and SARS-CoV-2 proteases are very similar with only some slight mutational differences (binding pocket is relatively conserved) so the drug made for CoV-1 can be used for CoV-2. The drug is a prodrug so that means the actual drug is an alcohol but they've put a phosphate group on that to make it much more water soluble. The phosphate group is cleaved off by the enzyme alkaline phosphatase and we get the actual drug which is a ketone based inhibitor that has an alcohol

Where is the substrate binding site of an enzyme?

The active site

How does competitive inhibition work?

The inhibitor looks just like the normal substrate and it binds in the enzyme active site so the actual substrate can't bind. It's also possible that the inhibitor may stay in there longer than the real substrate and just lock up that enzyme for a long time rather than letting it continue to do it's job.

What is the role of water?

The intermediate then reacts with water, which displaces the remaining part of the initial substrate and reforms the initial enzyme. Histidine thinks that the water looks like serine's hydroxyl and it grabs one of waters protons and creates an activated hydroxyl intermediate. This acts similarly to the alkoxide ion by making a nucleophilic attack on the carbon and then it's released and the enzyme regenerates.

Why do we have so many models?

There are so many different models because different enzymes seem to be better explained by different models.

What are restriction enzymes? Where do they come from?

They are enzymes that recognize and cut DNA at specific sequences. They cut a staggered/sticky end They come from bacteria and they're a bacterial defense mechanism

What puts those AAs next to each other (i.e., they are not all in a row in the primary sequence)?

They are put next to each other due to the folding of the protein

How much do enzymes enhance reaction rates?

They enhance reaction rates A LOT Enzymes accelerate the rates of such reactions by well over a million-fold, so reactions that would take years in the absence of catalysis can occur in fractions of seconds if catalyzed by the appropriate enzyme.

What does alpha-1-antitrypsin inhibit?

Trypsin and Elastase Elastase is in the lungs and plays a role in taking care of the lungs and keeping everything clean. Too much elastase is bad and can cause emphysema

What are the types of S1 binding pockets? What do they recognize?

Trypsin: Has a negatively charged bottom/ aspartic acid. It favors the positively charged AA of Lysine and Arginine Chymotrypsin: Broad, Nonpolar. The amino acids they recognize are phenylalanine, tryptophan, and tyrosine Elastase: Narrow, Nonpolar. The AA it recognizes are glycine, alanine, and valine

What is Kcat?

Turnover number. the constant of catalysis It doesn't vary w/ the amount of enzyme so you CAN compare Kcat values Vmax / [E used] = [P] / [E used] x Time

What is the MM equation?

V = Vmax[S] / Km+[S] Vmax: Occurs when an enzyme is saturated by substrate and varies w/ the amount of enzyme used Km: is a measure of an enzymes affinity for its substrate and is inversely related to affinity

What is the alkoxide ion? Where does it attack?

VERY reactive oxygen that's on Serine from the leftover acid/base catalysis An ion with a negative formal charge on oxygen atom bonded to an sp3 carbon atom Attacks the carbonyl group of the peptide bond of the substrate.

How do bacteria protect themselves from restriction enzyme cutting while at the same time using them to get rid of viral DNA?

Via Methylase The Bacteria puts methyl groups on DNA of that sequence that is the same as the one the restriction enzyme is going to cut. The methyl groups tell the restriction enzyme that it's not in a recognizable place to cut. However, these methylases can unfortunately methylate the viral DNA but usually the restriction enzyme gets in there first so the methylases doesn't have time to methylate. Restriction enzyme can also accidentally cur the DNA if they haven't been methylated so it's all about timing.

What happens if we mutate the DNA to change one of the AAs in the catalytic triad?

We can mutate by using site direction mutagenesis and it will make it so that codon will code for a different AA. The mutations destroy some of the activity for the enzyme. It makes the enzyme less effective. The more similar the mutation is to the original it has better activity/effectiveness but not like how it was before If the enzyme exists at all, it's better than no enzyme but not by much

Where is the steady-state phase? Why?

Where [E] and [ES] aren't changing/relatively constant so that you can measure the kinetics

How do we graph the rate of product formation over time?

X-axis: Time Y-axis: Concentration [] Then there are 4 lines: [S], [P], [E], [ES]

What is the laboratory use of staggered ends (or sticky ends)?

You can also put DNA that you're interested in there too. You can fuse it to other DNA, you can insert it into a plasmid that will artificially express a gene (sticky ends are great for that). You can engineer your piece that you want to put in to match with the overlap of the sticky ends.

Why is it good to do an enzyme kinetics graph for this type of assay rather than just looking at a single end point?

You don't want to look at just one point at time because if you do you don't know the exact amount of chymotrypsin. You add plenty of the molecule and the chymotrypsin is allowed to work for a long time and the rate levels off and you end up with the same amount of color at the end. You want to use enzyme kinetics because if will give you a more accurate idea of how much chymotrypsin you actually have. The reaction rate can be visualized. Another Justification: - Is a 2-step reaction. Burst phase and Slow Step. - If just looking at single end point, won't be able to see how much chymotrypsin you have b/c it's a 2-step reaction - Burst phase (fast): peptide bond is broken. Product is covalently attached to enzyme. 1 part released (yellow) - Slow phase: The product that attached to the enzyme needs to be released so it can grab more of the assay molecule.

What is an "active site"?

the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. It is where the reaction is catalyzed Substrates bind to the active site and they are often on clefts.

What is Km?

the concentration of substrate which permits the enzyme to achieve half Vmax.