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Bushra Ghnimat

Noor Shahwan

Name

Nafeth abu tarboush

26A

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Recap:

1- Binding leads to formation of transition state. 2- Usually, substrate binds by non-covalent interactions to the active site. 3- The catalyzed reaction takes place at the active site, usually in several steps.

How do enzymes work? (from a theoretical point of view)

Two models for enzyme-substrate interactions:

1- Lock and key model: this model adopt the idea of having 100% complementarity between the active site and the substrate.

The lock and key model was a previous model. It still can explain some of the enzymes

action, but it failed on explaining how do most of enzymes work, because of two

reasons:

1- With the development of biochemical techniques —> enzymes are proteins and proteins are not static in solutions. They are dynamic. They can change their conformation upon binding molecules , so accordingly if the active site is 100% fit to the substrate , it doesn’t make sense because enzymes change their structure , their shape even a little bit and accordingly the chemical structure of the substrate is rigid . So, it doesn’t make sense for a rigid thing to bind a dynamic thing in a key and lock model.

2- With time —> it appears that enzymes can have more than one substrate. For example, glucokinase binds glucose and binds ATP to transfer the phosphate from ATP to glucose and the product will be glucose six phosphate and ADP.

So, this active site has the ability of binding two different substrates. And even if this

makes sense. What doesn’t make sense according to the key and lock model that the

same enzyme can bind different substrates at different times. The same enzyme

glucokinase can bind glucose with ATP to make glucose six phosphate, but with a

different reaction, it can bind fructose with ATP to make fructose six phosphate.

If there is a lock, there should be one key to open it, it can’t be different Keyes.

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2- The induced fit model (the adopted theory now)

Which says as the name implies that 100 % complementarity in between the active

site and the substrate is induced ----> there is a degree of complementarity between

active site and substrate. After the initial binding of the substrate with the active site

——> the active site changes its shape and the substrate orients itself within the

active site to the best fit, so the best reaction can occur.

How do enzymes work? (from an energy point of view)

Enzymes decreases the amount of energy needed to overcome the energy barrier

(which should be there all the time even if it is small, so not all reactions will go

through spontaneously) and achieve the transition state.

The transition state is the most unstable state while going from reactants to

products.

Accordingly, if we decrease this energy barrier, the reaction can happen, and this

reaction barrier can be overcome easily ——> this is what enzymes do ——> they

decrease the activation energy.

➢ Enzymes speed up reactions but have no relation to equilibrium or favorability.

➢ The activation energy: energy difference between the reactants and the

transition state. ➢ Specificity varies (stereoisomers), however, there is none non-specific. ➢ The rate of a reaction is independent of its spontaneity.

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For sure the transition state is an intermediate state, so for some reactions we go through different intermediates. Which one of them is exactly the one needed and we call it the activation energy (which leads to the transition state of the molecule)?

The one with the highest energy.

Substrates of enzymatic reactions often undergo several transformations when associated with the enzyme and each form has its own free energy value.

Y axis: free energy

X axis: the progress of

the reaction (where we

go from point A

(reactants) to point B

(products)

the arrow indicates the

activation energy for the

uncatalyzed reaction to

convert the stable substrate

into an unstable transition

state (without the presence

of enzymes).

We have here 5

intermediates

activation energy for

catalyzed reaction (with

the presence of enzymes)

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This is a real example for an enzyme which is called adenosine deaminase.

How do enzymes work? (from a mechanism point of view)

According to random collision theory, the molecules keep moving in any solution

till they find each other, and they collide with each other. This collision will result in

energy until we reach a successful collision (which means the amount of energy out

of this collision will overcome the energy barrier) -----> which will lead to product

formation.

Proximity effect: bring substrate(s) and catalytic sites together. Explanation: we will save time by not letting molecules keep moving randomly in the solution by having an affinity of these molecules toward the specific place (active site). BY binding to them then they are within small area bound together, and they can bind each other. they can collide with each other. they can do reaction with each other, so we bring things together. IMAGINE if you want to meet your friends and the only information you are telling him is to meet him at the uni. Imagine how much time do you need to find him! you will never find him in a reasonable amount of time. But if you told him that you will meet him at the medical faculties, then the chance will be higher. if you said that you will meet him at the school of medicine, the chance will be higher and higher because you are making the area of looking for smaller and smaller. And if you told him that you will meet him at chair X in room X, then you will decrease the time to find him -----> and this is what enzymes do.

The symbol for

transition state

3 intermediates

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Orientation effect: hold substrate(s) at the exact distance and in the exact orientation necessary for the reaction. Explanation: the substrate after it binds the active site, it orients itself by the help of the active site, so as the best fit to be. IMAGINE if you have an active site lined by hydrophobic and hydrophilic polar and charged amino acids. The nonpolar amino acids will bind the nonpolar parts of the substrate, while the polar amino acids will start doing the catalysis. So, when the substrate binds, it will orient itself in the best way, so as the nonpolar part of the substrate will bind the nonpolar part of the active site.

Catalytic effect: Provide acidic, basic, or other types of groups required for catalysis.

Energy effect: Lower the energy barrier by inducing strain in bonds in the substrate molecule. Explanation: after the substrate is in the best orientation ----> The active site closes itself on the substrates -----> bringing the molecules close to each other ----> start binding the substrates from outside -----> weakening the bonds inside the substrates and accordingly some bonds will start breaking, some bonds will start reforming and the process will go through changing the energy level within these bonds ----> the catalysis process will occur.

Strategies for catalysis:

Catalysis by proximity & orientation

❖ Enzyme-substrate interactions orient reactive groups and bring them into proximity with one another favoring their participation in catalysis

PhenylalanineThis is

It fits itself inside the

active site in a

.hydrophobic pocket

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▪ Such arrangements have been termed near-attack conformations (NACs) (the active site puts the substrate in the nearest attack conformation) ▪ NACs are precursors to reaction transition states

Catalysis by bond strain

❖ In this form of catalysis, the induced structural rearrangements produce strained substrate bonds reducing the activation energy.

This is a strategy applied by the enzyme lysozyme where 3D shape of the monosaccharide

will be distorted from the typical 'chair' hexose ring into the 'sofa' conformation. HOW?

The active site will enclose the substrate ---- > apply a strain on the bonds to change the

angles --- > increasing the energy within them ---- > then it can break after.

Catalysis involving proton donors (acids) & acceptors (bases)

- The R groups act as donors or acceptors of protons

- Histidine is an excellent proton donor/acceptor at physiological pH

▪ Example: serine proteases.

This strategy involves having polar (acidic and basic) amino acids within the active site.

Accordingly, they can donate protons or accept them from the substrates.

If you take a proton from the substrate, the substrate will have less protons → it will be

unstable → then it will try to abstract a proton from elsewhere → the bond will be

broken.

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Covalent & ionic catalysis

- Ionic catalysis is the same as what we said about the acid base catalysis.

- Covalent catalysis

- sometimes enzymes cannot be active unless by breaking down of certain piece of that

enzyme.

In covalent catalysis, we can covalently catalyze the enzyme. For example, a lot of enzymes are produced in an inactive manner and then they will be active through covalent catalysis. WHAT DO WE MEAN BY THAT? Breaking down of certain piece of the enzyme, A piece will be closing the active site, close it, break it down → so the peptide bond will be broken and then this piece will be gone irreversibly and the active site is open again for the enzyme to do the catalysis . ▪ Lots of enzymes (which are preceded by Pro or followed by gen) like Pepsinogen

will be pepsin, Trypsinogen will be trypsin, prothrombin will be thrombin, Fibrinogen will be fibrin, work in this way.

▪ covalent catalysis enzyme is not active → by binding phosphate, it will be active and vice versa.

▪ There should be no initial covalent interaction between the active site and the substrate. Otherwise, we will consider this interaction as an irreversible inhibitor to the enzyme. A covalent bond might occur during the mechanism though, but it’ll be broken again.

❖ A covalent intermediate forms between the enzyme or coenzyme and the substrate

❖ Examples of this mechanism is proteolysis by serine proteases, which include digestive enzymes (trypsin, chymotrypsin, and elastase)

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Naming enzymes

❖ In general, enzymes end with the suffix (-ase) some enzymes inherently got their names like (proteolytic enzyme trypsin, pepsin,

chymotrypsin) and their names don’t give any information about their work

❖ Most enzymes are named for their substrates and for the type of reactions they catalyze, with the suffix “ase”added (first: the name of substrate followed by the type

of reaction then the suffix [ase])

few amounts of them are named according to their product like citrate synthase.

❖ For example, ATPase is an enzyme that breaks down ATP, whereas ATP synthase is an enzyme that synthesizes ATP (ATPase and ATP synthase are the same molecule, its

name differs according to the situation)

Naming of enzymes; EC numbering (enzyme commission

numbering)

❖ Most scientific method. however, it doesn’t lead you to any information while studying and it is confusing.

❖ A numerical classification scheme for enzymes, based on the chemical reactions they Catalyze

❖ Strictly speaking, EC numbers do not specify enzymes, but enzyme-catalyzed reactions

❖ Numbering format: - EC followed by four numbers separated by periods

-Major class (1-7), Minor class, subclass, further sub-classification.

❖ For example: tripeptide amino peptidases "EC 3.4.11.4” First digit is what we are interested in.

EC 3: hydrolases

EC3.4: hydrolases that act on peptide bonds

EC3.4.11: hydrolases that cleave off the amino terminal of the amino acid polypeptide

EC3.4.11.4: cleave off the amino-terminal end from a tripeptide

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Enzyme classification (structure)

- Simple → simple enzyme can do its action through the amino acids sequence alone

such as trypsin, pepsin.

- complex (conjugated) → complex enzyme should have coenzyme, cofactor

Or Metal (non proteineous compound) binds to the enzyme, so the complex enzyme

becomes active, such as catalase enzyme that uses heme group to break hydrogen

peroxide into water and oxygen ----> heme is a non-protein compound that has nothing

to do with amino acids (the enzyme is synthesized first, the heme is added and then the

enzyme will be active).

-Holoenzyme (enzyme with coenzyme) & apoenzyme (enzyme without having

coenzyme) regardless the substrate being there or not.

Enzyme classification (function) (major class from 1 to 7)

1- Oxidoreductases: addition or removal of O, O2, H & Require coenzymes(heme).

- Reduction: gain of electrons.

- Oxidation: loss of electrons.

They occur simultaneously, any oxidation should be accompanied by reduction, so oxidoreductases reaction should have at least 2 reactants and 2 products.

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.transfer of a group from one molecule to another :Transferases-2

- catalyze the transfer of functional group from molecule to another, so it should be at

least 2 reactants and 2 products in the reaction.

3-Hydrolases: addition of water (carbs. &proteins)

- Hydrolysis means using water to breakdown molecules which are made by release of

water molecules which involve all macromolecules (lipids, carbohydrates, nucleic acids,

proteins).

-addition of a molecule (H2O, CO2, NH3 to a double bond or reverse(non Lyases:-4

hydrolytic).

- catalyze the addition or removal of functional groups, but the reaction is coupled to

formation or breakdown of a double bond.

-Lyases can add water as hydrolases, but this reaction involves the breakdown of double

bond not the whole molecule (the product is one molecule).

(we add water to alkene to produce alcohol----> one carbon of the double bond takes

the OH group and the other takes hydrogen atom)

while hydrolases break down the whole molecule.

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cause isomeration reaction.> ---product one substrate and one Isomerases:-5

➢ mutases are a subtype of isomerases.

.not favorable, so they require a simultaneous hydrolysis reaction usually Ligases: -6

o As the name implies, they build up molecules to make bigger molecules. Accordingly, those reactions are not favorable (because they need energy for them to occur). ➢ The breakdown of a molecule >> releases energy. ➢ Building up of a molecule >> requires energy.

o With these reactions, we can find ATP (not as a source of phosphate only, also as a source of energy).

Transferase gives energy and at the same time it gives the phosphate to bind the substrate of the reaction

While in ligases ----> ATP serves as energy source for the reaction.

Catalyze the movement of ions or molecules across membranes or Translocases:-7

their separation within membranes (ATP/ADP translocase).

▪ They are enzymes within the membranes mainly and cause the translocation of material from outside to inside or from inside to outside or in both directions.

▪ They also can catalyze the breakdown of a substrate inside the enzyme Example: ATP/ADP translocase that translocate ATP from mitochondria (the factory of

ATP) to the cytosol to build up molecules and translocate ADP to inside the

mitochondria.

つづく

We can see CO2 binds the pyruvate (which is a 3-carbon molecule) to make 4 carbon

molecule --> ATP serves as a source of energy to release ADP and inorganic

phosphate in the product.