13 14 Biochemistry Elliot Enzymes 1-8-14

8/13/2019 13 14 Biochemistry Elliot Enzymes 1-8-14

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Biochemistry

Elliot

January 8, 2014

10:00pm-11:00am

NT #5

Enzymes

Slide 21: Next, weregoing to talk about lysozymes and how they destroy cells.

Slide 22:This slide shows a lysozyme (left) and a peptidoglycan (right). What we are doing is trying to cut

the bond (beta-1,4 linkages--red arrow) between the two sugar residues (yellow dots) and if we cut

enough of those beta-1,4 linkages, the integrity of this layer is lost and the bacteria is lysed. The amino

acid sequence is shown below the picture of the enzyme structure. The amino acids marked in the

sequence are the ones whose side chains participate in the active binding of the substrate to form the

enzyme-substrate complex.

Slide 23:This slide has a different view of the same enzyme with a space filling model. There is a

substrate bound across a very deep cleft in this molecule. When you do that, you get a bend in the

active site which torques one of the beta-1,4 linkages, causing it to shift from a boat conformation to a

chair conformation which is highly reactive. The rest of the substrate is still very stable, but at the active

site we end up with one strained bond.

Slide 24:When you strain that bond in the chair conformation you end up putting a partial charge on

the oxygen in the substrate in the active site. This oxygen is immediately adjacent to glutamic acid that

is protonated. This is unusual to have a protonated glutamic acid but because it is found in the

hydrophobic domain, it doesnt want to possess a charge. This causes its pKa to shift so that it holds

onto that proton. Now when the substrate comes in and brings the strained partially negative oxygen

next to the protonated glutamic acid, the glutamic acid ends up donating its proton to the oxygen of the


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substrate. This sets up a partial positive charge on C1 of the sugar ring. That partial positive charge of

the carbocation is immediately adjacent to an ionized aspartic acid side chain in a hydrophilic domain on

the opposite side of the substrate binding cleft. The negative charge on the oxygen goes to the positive

charge on this carbon and creates a transient covalent intermediate. At this point water comes back in

as the E-strand diffuses away. Meanwhile, the glutamic acid is still negatively charge but wants to be

neutral since it exists in a hydrophobic domain. Therefore the glutamic acid takes a hydrogen from the

water to become neutral again, leaving a hydroxyl group. This hydroxyl group then attaches to the acid-

anhydride linkage, and attacks the partially positive charge of the carbon, releasing the aspartic acid of

the enzyme back to the original conformation. You form a transient covalent bond and then reverse

everything back to the original conformation using water. This is a nice simple, direct acid-base

hydrolysis reaction. We do not need to verbatim memorize this reaction for the exam. However, we

should understand how a lysozyme works.

Slide 25:These videos are included to help us understand the process lysozymes use. He did not show

these movies in class, but they will re-explain the reaction he just went over. The first one is more

general, and the second one goes over the reaction mechanism.

Slide 26:This is an example of a serine protease. Chymotrypsin is a digestive enzyme kicked out by thepancreas into the small intestine that is used to digest dietary proteins that have been denatured by

stomach acids and partially cleaved by pepsin. He is showing a serine protease because there are a lot

of these that have clinical relevance such as digestive enzymes. The entire clotting cascade is a series of

serine proteases. The acetylcholine esterase is very similar to and derived from a serine protease so

nerve conduction also uses a similar mechanism.

Slide 27:Chymotrypsin hydrolyzes peptide bonds. When you hydrolyze a peptide bond, you have a new

carboxyl terminus and a new amino terminus. This slide shows an example of what chymotrypsin does:

it hydrolyzes a peptide bond, but it is specific. It doesnt hydrolyze all of the peptide bondsit only

hydrolyzes the bond on the c-terminal side of hydrophobic amino acids. In this case we are looking at

phenylalanine. Methionine is also shown, and this would work on leucine, isoleucine, and the aromaticsas well. Other proteases have different specificity based on the composition of their active site. Note

that chymotrypsin is not made in its active form so that it doesnt digest the organ that makes it. This

makes it a zymogen. Usually this means it has an extra polypeptide chain and is proteolytically activated

by trypsin (which is also a serine protease).

Slide 28:This slide shows chymotrypsin after its been catalytically reorganized. If you look in the yellow

circle, this is where the catalytic triad exists. This is a charge relay network, meaning that as the reaction

takes place, a charge is generated and then stabilized by another charge nearby. This allows us to get to

that high energy transition intermediate. This shows the active site with a binding cleft that contains a

serine, a histidine, and an aspartic acid. The serine has a hydroxyl group on its side chain that does the

reaction chemistry. The histidine next to it picks up a proton from the serine, giving the histidine a

positive charge. The positive charge on the histidine is then stabilized by the negatively charged aspartic

acid.


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Slide 29:This slide shows what the active site looks like without the rest of the protein surrounding it.

The serine wants the proton so much that is strains in order to reach the hydrogen. This generates a

partial positive charge in the histidine that is stabilized by the negative charge of the aspartic acid.

During this reaction, an oxyanion is generated which fits into the oxyanion hole which is immediately

adjacent to the active site. The negative charge on the oxyanion is stabilized from two hydrogen bonds

that come from within the backbone of 2 glycines. This is part of the stabilization of the transient

intermediate, and this part is common to all serine proteases.

Slide 30:This shows the reaction mechanism. We need to know the medical significance of serine

proteases (ie what happens if they get stuck in the duct) but also the kind of reaction this is (ie transient

covalent interactions). We dont need to learn the entire reaction. Here is how the mechanism works:

Here is the substrate that binds to the binding pocket and presents a peptide bond that is immediately

adjacent to a hydrophobic amino acid. The strained oxygen that is partially negative sees back side of a

carbonyl group which has a partially positive carbon. The carbon and oxygen form an ester-like linkage.

The hydrogen from the serine side chain is donated to the histidine, generating a partial positive charge

that is stabilized by the negative charge on the aspartic acid. The aspartic acid is what allows this

reaction to proceed so if the aspartic acid is mutated, the reaction does not occur. The proton that

serine gave to histidine is now donated to the new N- terminal group, causing it to diffuse away. Now,

because you have lost the partial positive charge on the histidine you have an unstable system. When

water comes in contact with the system, the negative charge from the histidine polarizes the water andtakes a proton, much like what we saw with lysozyme. The remaining hydroxyl group attacks the carbon

in the ester linkage formed earlier. This breaks the bond with the serine oxygen. The serine oxygen takes

the hydrogen back from histidine. This releases the new C-terminal end. The negative charge on the

carboxyl group on the new C-terminal and the negative charge on the carboxyl group of aspartic acid

repel each other and this helps eject new C-terminal from the active site. At this point everything has

been reset to its original form and the reaction can occur again.


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This is a two part reaction mechanism with a covalent intermediate. The first part of the

reaction is cutting the peptide bond and this happens very quickly. The second part of the reaction is

regenerating the enzyme by hydrolyzing water. The water component is the rate limiting step (RLS) in

this mechanism because it takes a while to get water oriented correctly. We will hear about RLS a lot in

this course, especially during reaction mechanisms and pathways.

Slide 31:This slide shows why you have substrate specificity. This shows the same reaction as the

previous slide. You can once again see the serine, histidine, and aspartic acid relay network where we

bring a polypeptide chain. Since the polypeptide has already been through digestive track, it is fairly

denatured. Immediately adjacent to the relay network where the polypeptide bonds, is a hydrophobic

binding pocket. This is a deep pocket lined by hydrophobic amino acids, meaning that the side chains

that fit into the binding pocket are also hydrophobic (eg leucine, isoleucine, etc). This gives the enzyme

the specificity to only cut peptide bonds that are adjacent to big, bulky, hydrophobic amino acid side

chains with chymotrypsin.

Slide 32:What about other enzymes? The reaction mechanism is the same in trypsin, elastin, and some

collagenases. However, in trypsin the binding pocket has an aspartic acid sitting at the bottom of it. This

means that positive side chains are attracted to it (this would be the basic side chains: arginine andlysine. Not necessarily histidine because we dont know if it will be protonated or not, but it could be).

Therefore trypsin cuts the peptide bond on the C-side of basic side chains. In elastase, there is a shallow

binding pocket so smaller side chains like glycine, alanine, which are abundant in elastin. So you see the

binding pockets determine the specificity, but the reaction mechanism stays the same for all of these

enzymes (serine, histidine, and aspartic acid are used to break the peptide bond, then hydrolyzed by

water).

Slide 33:Now well go back to kinetics. We do not need to know how to derive this equation but we

should know what it is and what it tells us so we can apply it.

Slide 34:This is the Michaelis-Menten equation.

Slide 35:If you measure the rate versus the substrate concentrations, you generally see this curve for all

enzymes called Michaelis-Menton enzymes. There are two major aspects of these curves that we can

use as rules of thumb for this class: Vmax is the max velocity at a given enzyme concentration (also fixed

conditions: temp, salt, pH etc). If you change any of the conditions then the constants (Vmax and Km)

would change. We cant go any faster than Vmax because we have saturated all of the substrate active

sites. At Vmax, where you intercept the substrate curve, this where we can find a gross approximation

of Km. This is only an approximation because our Vmax is actually an approximation (since we dont

bring the chart out to infinity.) If Vmax is an approximation, then Vmax has to be an approximation as

well. Notice that because Km intercepts on the substrate axis so it has substrate units (units of

concentration). For example, if Km is 10mM, then when the substrate is at 10mM then we know that

the reaction is rate is at of Vmax.


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Slide 36:[Example not included on slide:] Hexokinase and glucokinase both phosphorylate glucose to

trap it inside the cell. Hexokinase phosphorylates other sugars as well so it is much less specific. The Km

for hexokinase is 0.1mM. The Km for glucokinase is 5 mM. That is a 50 fold difference! Blood glucose

concentrations are usually 4-6 mM. Because hexokinase has a Km of .1 mmol, blood glucose is already at

saturation of that enzyme. That means that glucose is immediately taken up by hexokinase when blood

comes in contact with muscle cell membranes. However, the Km for glucokinase is 5mM. This Km is so

much higher because the liver doesnt want to trap all of the glucose that it comes in contact with so

that the brain is able to obtain enough glucose. When there is excess blood glucose, the liver will store

the excess glucose because of its Km, but if blood glucose levels are low, then the liver wont take up the

blood glucose.

This slide shows the actual equation. Hell put example problems for this on blackboard. Werelooking at the maximum velocity determined from the total concentration of active sites. Km can be

looked at as the dissociation constant over the association constant. It is sort of like a binding affinity

value.

Slide 37: Here are some examples of Km, and some problems we might run into when looking at these

values. You can see that lysozyme has a very high affinity for its substrate with a Km of 6uM. Notice that

carbonic anhydrase has a Km of 8000mM. This is deceptive and means that you do need to have an

understanding of what is happening with the forward kinetic constant. If that constant is a huge

number, then it will arbitrarily make Km look like a huge number.

Slide 38:One of the things we look at to get a better sense of what is going on with Km is Turnover

Number. The turnover number is equal to the maximum velocity divided by the total concentration of

enzyme active sites. This is equal to the number of reactions per unit time or how fast you can turn a

substrate into a product. For example, lysozymes can turnover 1 time every 2 seconds, whereas carbonic

anhydrase can turn over 600,000 times per second. A very efficient enzyme like carbonic anhydrase

almost instantly runs the reaction. Lysosymes are very efficient, but have a much larger substrate that

takes longer to bind.


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Slide 39:Essentially some of the reactions are so perfect that they occur at the speed of diffusion.

When kcat (forward kinetic constant) is very large, if you divide kcat by Km, the whole equation reduces

to k1. k1 is a measure of how well the substrate associates with the enzyme from the enzyme substrate

complex. The second it touches the enzyme substrate complex it reacts. This can make Km for perfect

enzymes look very large. However this isnt a real determinate of binding affinity. Instead, that is the

limit of diffusion. You cant go any faster than diffusion. So if you see a k1 is in the range of 10^7 or

greater, this means that the enzyme has evolved to the point that the second the substrate touches the

enzyme, the reaction takes place. That is rare, but includes things like acetylcholine esterase and

carbonic anhydride.

Slide 42:Now well talk about irreversible inhibition and suicide inhibition. For our purposes, these two

are the same thing. In technical terms these are not the same thing because in suicide inhibition, the

substrate creates the enzyme that irreversibly inhibits it. These inhibitors are both compounds that bind

with high affinity to the enzyme, inhibit its activity, and fail to release.

Slide 43:Aspirin is the classic example. Aspirin usually has a short half-life. However, if aspirin binds to a

specific enzyme, in this case prostaglandin synthase, it will fold up. What will happen is that these

prostaglandin synthase enzymes bind to aspirin, which then acetylates a serine in the active site of theenzyme. This wipes out the enzymes activity because serine is part of its reaction mechanism. This is

damaging to the stomach lining because prostaglandins help promote cell growth that is important for

the stomach lining which regenerates every 2-3 days. This is why its better to use a coated aspirin that

wont be digested until it reaches the small intestine (rather than the stomach) or use take a newer drug

similar to aspirin that has more specificity, such as Celebrex that targets COX2 only rather than both

COX1 and COX2.

Slide 44:Acetylcholine esterase is an enzyme that destroys acetylcholine. It sits in the meshwork in the

synaptic cleft. The Km for this enzyme is huge because it is a kinetically perfect enzyme. When you

release acetylcholine into a synaptic cleft, most of it is broken down as it passes through the cleft.

However, because of the relatively large amount of acetylcholine released during an intentionalmovement, enough makes it to the other side to bind to the post synaptic element.

Slide 45:When acetylcholine esterase binds ACh, there is an anionic site that binds to the choline

moiety. The acetate binds into a serine based active site. Then there is hydrolysis of the alcoholic bonds

and choline is released. The acetic acid is covalently bound to serine. And then water comes in and

hydrolyzes the acetic acid off to regenerate the active site. This is another example of transient covalent

intermediates that are released by water hydrolysis. This is shown by number 1.

In the upper right corner you can see two neurotoxins that affect acetylcholine esterase: Sarin

and VX. These nerve agents show their organophosphates. These organophosphates form a covalent

bond between the hydroxyl group (because fluorine is a great leaving group) and the serine, shown in 3.

This bond is somewhat unstable so it reorganizes itself into a more stable bond shown in 4. The bond

shown in 4 is a very stable bond that will never come off. This is the irreversible binding of the inhibitor.

The effect of this is that ACh is no longer broken down, causing respiratory arrest because the

diaphragm can no longer relax.

Slide 46:Pyridine Aldoxime Methiodide (PAM) and Atropine are used to protect against these

neurotoxins. The PAM binds to the choline hydrophobic pocket. The hydroxyl amine group binds to the

phosphate. It breaks a bond between the organophosphate and the serine and forms a hydroxyl amine.

Once it forms that bond the neurotoxin compound releases (top molecule in the bottom pic). The


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cytochromoxidases in the liver is able to break these toxins down so they can be passed out of the

system (although the liver doesnt like this). Also the atropine competes with the ACh as the receptor

and blocks the interaction of ACh with its receptor in order to block the paralysis effect.

Slide 47-50:Penicillin is a transition state analogue. He didnt cover penicillin in classhe just wants us

to read what is in the slide notes.

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13 14 Biochemistry Elliot Enzymes 1-8-14