Enzymes, Catalysis, and Serine Proteases

Roadmap for Lectures

• Upcoming Topics:
  • Finish Chapter 8 (first lecture set).
  • Finish hearing about proteases.
  • Start Chapter 9 (monosaccharides, simple sugars).
• Preparation for Wednesday's Class:
  • Watch the first lecture video for Chapter 9.
  • The lecture will cover material from the video but will include additional insights, hints, and tips.
• Exam Preparation:
  • Chapter 9 will involve memorizing structures.
  • This is similar to memorizing structures for reactions.
  • Memorization will be relevant for the next exam.

Catalysis

• Acid-Base Catalysis:
  • Amino acids can donate/accept protons.
  • This makes functional groups more reactive.
• Covalent Catalysis:
  • Covalent bonds form between enzyme amino acids and substrates.
  • This changes the reaction pathway compared to uncatalyzed reactions.
  • Additional steps may be added, but each step has a lower activation energy.

Example: Decarboxylation of $\beta$-keto acids

• Spontaneous Decarboxylation:
  • Elacetate forms CO2.
  • Forms a high-energy enolate transition state.
  • The high-energy transition state makes the reaction slow.

Tweaking the system

• Nucleophilic Attack:
  • Deprotonated lysine side chains are common nucleophiles.
  • These processes often have high activation energy.
• Goal:
  • Achieve more favorable, lower-energy transition states via covalent catalysis.

Imine/Schiff Bases

• Importance:
  • Important in biochemistry, especially metabolism.
  • Involve carbonyls interacting with amines.
• Mechanism:
  • Not responsible for arrow-pushing or fine details.
  • Key is to understand that it changes intermediates and transition states.
• Process:
  • Carbonyl interacts with amine.
  • Acid-base catalysis (protonation/deprotonation events) is involved.
  • Oxygen leaves as water.
  • Forms a carbanol double bonded to a nitrogen (imine/Schiff base).
• Electronic Changes:
  • Carbonyl: Polar (partial negative on oxygen, partial positive on carbon).
  • Imine: Positive charge on nitrogen.
  • The electronic change on the carbon makes it more reactive.
• Transition States:
  • Imine formation leads to more stable transition states compared to enolates.
• Reversibility:
  • The reaction must be freely reversible.
  • Water is added back to return to the starting materials.

Stabilization

• Carbanion Stabilization:
  • The positive charge on the nitrogen stabilizes the carbanion.
  • This leads to lower activation energies of transition states.
• Enzyme Catalyzed Reaction
  • Uncatalyzed Reaction: Very high-energy transition state.
  • Lysine Side Chain: A series of events occurs, resulting in the formation of an indium.
  • Electron Density: The partial positive charge on the carbon, as well as the partical negative charge on the the oxygen, the electron density is strongly pulled towards the nitrogen as electrons have a negative charge, opposites atract and pull the electrons in that direction.
• Comparison:
  • Carbonyl (partial positive on carbon, partial negative on oxygen).
  • Imine (full positive charge on nitrogen).
  • The carbon in the imine is more electron deficient.
  • This makes the carbon more reactive.
• Decarboxylation:
  • A neutral transition state is more favorable than a charged oxygen (enolate).
• Resonance Form:
  • Negative charge carbanion adjacent to positive charge nitrogen.
  • Still high energy but more stabilized.
• Overall Effect:
  • Lowers activation energy.
  • Lowers energy of transition states.
  • The reaction becomes more favorable.
• Final Step:
  • Water attacks to reverse the imine/Schiff base formation.
  • This regenerates the product and regenerates the starting material

Covalent Catalysis

• Goal:
  • Make centers more reactive.
  • Change electronics.
  • Lower energy of transition states.
  • Achieve a faster rate.
• Understanding the Concepts:
  • Not responsible for knowing every detail of what's going on.
• Side Chains:
  • Various side chains can participate in covalent catalysis: deprotonated alcohols, thiols, adenines, histidines.
• Serine Proteases:
  • Good example of covalent, acid-base catalysis, and transition state stabilization.

Metal Ion Catalysis

• Definition:
  • Any catalysis involving metal ions.
  • Can fit into previous categories (proximity, orientation, transition state stabilization, etc.).
• Functions:
  • Help align substrates, intermediates, transition states.
  • Help with proximity and orientation.
  • Undergo oxidation-reduction reactions.
• Water Activation:
  • Metal ions can interact with water molecules to make them more reactive.
  • Hydroxide (deprotonated water) is a strong nucleophile.
  • Metal ions can facilitate acid-base catalysis.

Metal Ion Functions

• Substrate Binding:
  • Help bind substrates, intermediates, and transition states.
  • Facilitate proximity orientation reduction.
• Redox Reactions:
  • Metals undergo oxidation-reduction reactions during catalysis.
  • Example: Enzymes converting proline to hydroxyproline and lysine to l-lysine.
  • Metal must return to original oxidation state.
• Enzyme Inactivation:
  • Enzymes can get trapped in wrong oxidation states.
  • This leads to a non-functional, inactive enzyme.
• Vitamin C Role:
  • Vitamin C can help regenerate the active enzyme by returning the metal to its reduced form.

Metal Ion and Water

• Coordination:
  • Metal ion coordinates to a neutral water molecule.
  • Oxygen coordinated to metal gains a positive charge.
  • Facilitates proton loss, forming a hydroxide coordinated to the metal.
• Example: Carbonic Anhydrase
  • Catalyzes the combination of water and CO2 to form a proton and bicarbonate.
  • Part of the blood buffering system.
  • Converts metabolic CO2 to dicarbonate.
• Active Site:
  • Contains catalytic zinc coordinated to histidines.
  • Zinc also coordinates to water.
  • Water needs to lose a proton to become more nucleophilic.
  • Hydroxide is a better nucleophile to attack CO2.
    Histidine: Is literally facing in where the water it sits, so is in the perfect location to extract a proton, folding funnel.
• Mechanism:
  • Zinc binds water, simplifying it.
  • Water deprotonates for nucleophilic attack.
  • Histidine acts as a proton pump, extracting a proton.
  • Protonation of histidine leads to a conformational change.
  • Bicarbonate leaves, and a new water molecule enters.

Carbonic Anhydrase Detailed Mechanism

• Enzyme Structure
  • crevice containing zinc coordinated to histidines.
  • CO2 molecule located near the active site.
• Water Molecule and Hydroxide Location
  • The water that is going to attack the molecule in the form of hydroxide is not even that close to the CO2 molecule, which seems counter intuitive.
• Activated Base
  • Histidine needs to be a lot more strong of base for it pull a proton to pull out the water molecules.
• Role of Multiple Water Molecules:
  • A series of water molecules fill the gap.
  • Each stabilizes the other by grabbing protons.
  • Forms a proton chain.
  • Histidine pulls off the proton, leading to hydroxide formation.

Metal Coordination Review (metal, water, histidine)

• Histidine's Role
  • The histidine now grabs a proton from the chain.
• **Histidine Releases a Hydroxide **
  • The water releases a hydroxide, which can then attack the carbonyl and give you now a bicarbonate. The thing is can't stop there as histamine is now protonated and is facing out and into a different environment, this allows the histamine to let go for it to become deprotonated once again, being ready to grab another proton.
• Proton Movement:
  • The Histidine literally keeps a proton pump and makes sure all hydroxides molecules can atack the CO2.
• Distance
  • The activated base in a hydroxide molecule that does the work will always have a distance to the proton, is a very similar thing as the metal itself in the slide.
• Three Main Functions of Metal Ions in Catalysis:
  • Metal ions help stabilize binding events with substrates, intermediates, or transition states.
  • Metal ions faciliate oxidation-reduction reactions, so in the process in transfering of electrons and and doing whatever chemistry has metal ions changing state of oxidation of state to help move those electrons effectively.
  • Metal ions can also coordinate to water and allow them to better leave when doing a catalytic action.

Serine Proteases Overview

• Definition:
  • A class of enzymes (not a single enzyme) that have been extensively studied.
  • A large focus has been placed on catalytic activity.
• Significance:
  • Demonstrates four out of the five classes.
• Hydrolyze
  • Hydrolyze Amy by Box.
• ** Proteases**: hydrolyzing proteins
  • RNA: hydrolyzing RNA
  • DNA ase: hydrolyzing DNA
• Catalytically active serine plays a key role in chemistry
  • Very important and place is a major role.
• If you want to destroy Hydrolyze and Amide bond take the sample and do the following
  • have the sample and contain it with an Amid Bond, boil it inside of six molar hydrochloric acid.
• They are able to cleave amyl bonds at temperatures close to normal human body temperature of 37°C
  • They also do it at a almost normal pH, 7 not 6 Molar Hydrochloric acid
• Selectivity
  • They are Selective and don't just cleaves single amide bonds in the protein, as we talk about how it's going to cleave different proteins in this family and how it's made up of three different proteins that are specific and are selective to certin AMID bonds.
    In order from any of the nutrients that we ingest, they have to be broken down and the smallest segment of a protein can be absorbed into intestinal cells is no larger then a tripeptide
• What do the serine porteases do
  • The ones we're going to talk about are digestive enzymes that help with the breakdown of specific amine-like bonds.
• 3 three serine protease enzymes
  • are trypsin, kimotrypsin, and elastase.

Serine Protease Preferences

• Chymotrypsin cleaves C-terminal to phenylalanine, tryptophan, and tyrosine.
• Trypsin cleaves C-terminal to lysine and arginine.
• Elastase cleaves C-terminal to alanine. The pocket is much smaller, so a large functional group won't fit in this Elastase.
  They cleave different bonds, but they do it in the exact same way. The difference is and what they recognize is a valid substrate, what I recognize is a valid bond, what I mean by by is good. You don't need 5, 6 or 7 different amino acids, one amino acid is enough for this to act.

Catalysis and Catalytic Center

• The immonotrubimus is coming in.
• And it doesn't matter which of these I'm doing. The immunotermis is coming in into this amino acid.
  There are permeases that are a lot more significant, the only thing that really matters for what's in the binding pocket, and in general only 1 bond is needed to activate the catalytic center.

Full Catalytic Cycle

The serium crow is very terriyfing, but it is actually only four steps that repeat a second time like nature does, its repeating the same code but twiking one of the parameters. it is a nucelophilic tactic carbonyl center from real attacking into a transition state, reforming the carbonyl, breaking a bond, having a nucleophilic. That's the first part. The second part is nucleophilic attack in the carbonyl center, making a centrifugal transition to reform the carbonyl, having a good leading period that breaks the bond.

Catalytic Triad

• Definition: The catalytic triad does the catalysis, the catalysis is made up of three amino acids. Aka, the catalytic triad.
• The amino acids that are going to do all of the fun chemistry are A aspartate, a histidine, and a serine, and it's the catalytic triad.
  Important: There is not an aspartate and histidine and a serine just floating around in space. These are actually part of the protein. Not shown here is the whole rest of the structure. This is just showing you that these three amino acids are near each other in three-dimensional space in the folded structure of the protein.
  Numbers and Primary Sequence, these nnumbers tell what amino acids are located and what number, so if you see numbers on there you can use those to help figure out what amino acid they are talking about.
• If these three things were not next to each other, shit would work and it's even more than that. It's not just to mirror each other. They have to be perfectly geometrically aligned for them to do the job that they're going to do. And so the catalytic triad is able to do its job only because of the shape of the molecule, because it folded properly to line these three things up perfectly.
  Alcohols have usually a PKA value of 14 to 16 so for this to work they should lose its proton. because standard Alcohols like searing us normally have a pka value of 14 to 16 so far out of the fiscal range she cares
  There is called a real special term and it is called a strong hydrogen bond, you can't exactly tell who owns it for it is equally distributed with both oxygen and the nitrogen.The important part is what base its strongest there so this process can accure.And This is showing you sort of where I start when everything is sort of in their expected state under physiological creation.
• When will this class. the oxygen on the Serine is going to be viewing are valent Calis is. The heating is going to be doing our acid-base Calis is. So that's kind of where we're heading with all this. and this is just kind of showing you, again, that perfect orientation of all the various things that will allow this to happen. But the catalytic triad is, of course, shared and is the same in all three of the serine proteases we talked about, or we're talking about elastase trips, and it kind of trips.

Divergent vs Convergent

• Divergent evolution is when nature arrives on a solution a problem and then that original protein over evolution changes and mutates to do other functions, but coming from a common ancestor. And so there might be other proteins that do new and different things, but they all originated from the same common solution, from the same original parent's protein.
• Convergent evolution is that if there's a really useful solution to a problem nature will find it in. a variety of different things. So not from the common ancestor branching out, rather from disparate different possibilities eventually getting to the same solution. The key factor here is that for it to work it must have those 3 factors 3d aligned.
  So this catalytic triad is a wonderful example of convergent evolution, where there is a great solution to a problem. In this case, how do you get an active nucleophilic oxygen? And nature lives at it in three different ways.

Catalytic Process Initiation

• The aspertates role basically makes the histamine a more better base than its protonated verison (base mode activated?)
  So even before we get into substrate bonding, we've already got some asymptomatic analysis going on. In this case, the histamine is serving with the base, removing the proton from the serine make it as strong as you would find. Getting to that idea that, you know, the aspartates role is to make the histamine a strong enough base that can actually distract the proton from the serine, lowering the effective pKa value of the serine enough that this can happen under visualizing conditions. And then this would be a good nucleophile to attack things.

Substrate and Catalyst Interaction

• R1 group is the side chain that fits in the selectivity pocket. So if we're doing kind of trips in, this would either be a, this would either be a tyrosine, or a tripped fan, or a thermal chain, all fitting those perfect spots of all the catalyst.
  What matters is that amine bond, and only this amine bond is going to be right next to that histidine that is going to pull a base, or sorry, pull a proton off of the oxygen on the serine to give me a strong oxygen nucleophile, an alkoxide, whatever you want to call it, an oxygen ion, that once it becomes incriminating, it can nucleophilic the attack at the carbonyl center. This is where the proximity orientation actually should begin. It is not a nucleophile.
  This deperonates, serves the base, we have the nucleophilic attack, the carbonyl center. When you leave the only fact that oxygen moves to a completely different position, at least now you can see those two hydrant bonds sort of stabilize.