“Introduction & Enzymes” “Introduction about Biochemistry & Enzymes”

Prerequisite terms

Electronegativity (EN): It is the measures of the tendency of an atom to attract a shared pair of

electrons (or electron density).

Covalent bonds can be classified as:

1. Nonpolar covalent bond (∆EN = 0 - 0.5), examples: C − C, C − H.

2. Polar covalent bond (∆EN = 0.5 - 1.9), examples: O – H, N – H.

❖ A polar bond has a negative end and a positive end.

❖ If ∆EN is more than 1.9, then the bond is ionic. Example: Li – F.

Also, they are classified as:

Bond Type Single Bond (–) Double Bond (=)

No. of Sigma Bonds 1 1

No. of Pi Bonds 0 1

Non-covalent interactions are (Backbone of the molecule):

1. Electrostatic interactions.

2. Hydrogen bonds (donor and acceptor).

3. Van der Waals interactions.

4. Hydrophobic interactions.

Hydrophobic vs. Hydrophilic Molecules:

❖ Something defined as hydrophilic is attracted to water.

❖ Something that is hydrophobic resists water.

Polar reagents can be divided into: Nucleophiles & Electrophiles.

1. A nucleophile has: A negative charge, a lone pair, or a π bond.

2. An electrophile has: A positive charge, a partial positive charge, or an incomplete octet.

Proteins are 1 or more polypeptide chain.

The polypeptide is several amino acids linked (held) by covalent bond called peptide bonds.

Amino acids are the monomers of all proteins.

There are 20 naturally occurring amino acids. The general form of amino acids is:

The R group determines the properties of the amino acid.

Peptide linkage: A bond formed between −COOH of one amino acid & −NH2 of another amino acid by

the removing of H2O.

Levels of protein structures:

1. Primary Structure:

❖ It is a linear sequence of amino acids in the polypeptide chain.

❖ It is the most important level of protein structure, and the simplest level.

❖ Peptide bond is the only bond that maintain (stabilize) the primary structure.

❖ The structure has 2 ends: N terminus & C terminus.

2. Secondary Structure:

❖ Coiling & bending into helices (α-helices) and / or sheets (β-pleated sheet).

❖ The bonds that stabilize the secondary structures are: Peptide bond (Covalent) &

Hydrogen bond (Non-covalent).

3. Tertiary Structure:

❖ Further (more) folding of the polypeptide upon itself to give 3D shape.

❖ The bonds that stabilize the tertiary structures are: Peptide bond (Covalent), Hydrogen

bond (Non-covalent), Ionic bond (Non-covalent), Hydrophobic interaction (Non-

covalent) & Disulfide linkage (Covalent).

❖ The last 4 bonds are formed between the R groups of the amino acids.

4. Quaternary Structure:

❖ Association (Union) of 2 or more polypeptides.

❖ The bonds that stabilize this structure are the same as the tertiary structure.

Biochemistry = Understanding life

Know the chemical structures of biological molecules.

Understand the biological function of these molecules.

Understand interaction and organization of different

molecules within individual cells and whole biological

systems.

Understand bioenergetics (the study of energy flow in cells).

Enzymes (Introduction)

General properties of proteins

The function of proteins depends on their ability to bind other molecules

(ligands, cofactors, substrates, etc.).

❖ Ligand: a substance that forms a complex with a biomolecule, usually

via non-covalent interactions, to serve a biological purpose.

Two properties of a protein characterize its interaction with ligands:

❖ Affinity: the strength of binding between a protein and other molecules.

❖ Specificity: the ability of a protein to bind one molecule in preference to

other molecules.

What are enzymes?

Enzymes: Specialized proteins that can conduct (catalyze) chemical reactions under biological

conditions. ❖ Exception: ribozymes (They are not protein; they are RNA molecules).

Most enzymes have very specific functions converting specific substrates to the corresponding

products. (Substrate to enzyme ratio is high, there is more substrates than enzymes).

Enzymes are catalysts.

❖ They exist in little amounts relative to

the reactants.

❖ They modify and increase the rate of a

reaction.

❖ At the end of the reaction, they undergo

no change.

Biochemistry in medicine:

1. Explains all disciplines.

2. diagnose and monitor

diseases.

3. design drugs (new antibiotics,

chemotherapy agents).

4. understand the molecular

bases of diseases.

How do we express an enzymatic reaction?

In enzymatic reactions: Substrates → Products.

Simple expression of enzymatic reaction:

E + S → ES → EP → E + P For simplicity: E + S → ES → E + P

E = free enzyme; S = free substrate, ES = enzyme-substrate complex; P = product of the reaction;

and EP = enzyme-product complex before the product is released.

***Note: The substrate changes its shape to form the products. The enzyme changes in the

transition state, then goes back to the original shape.

What do enzymes do?

Enzymes accelerate reactions (usually within a range of 106 to 1014 -up to 1020).

❖ Example: Catalase (108) & carbonic anhydrase (107).

Carbonic anhydrase:

One enzyme molecule hydrates 106 molecules of CO2 per

second (versus 1 per 10 seconds for uncatalyzed reactions).

***Note: Platinum surface can increase the reaction rate

because it brings the substrates together, increasing the

probability of collision, catalyzing the reaction.

Where does the reaction occur?

Each enzyme has a specific three-dimensional shape called the active site (a region where the

biochemical reaction takes place).

The active site contains a specialized amino acid sequence that facilitates the reaction.

Binding of a substrate into the active site can be regulated by a regulatory site.

Catalytic group

Within the active site are two sub-sites, the binding site, and the catalytic site.

The catalytic site contains residues (catalytic group) that carry out the actual reaction.

In some enzymes, the binding and catalytic sites are the same.

Examples:

❖ Picture 1 showing that the person is using his two hands to accomplish the task (It is like

the enzymes with binding & catalytic sites).

❖ In picture 2, the girl is using only one hand to accomplish the task (It is like the enzymes

that the binding & catalytic sites are the same).

Binding specificity

The specificity and selectivity of enzymes is due to their precise interaction of active sites to their

substrates and the degree of compatibility for this interaction.

***Notes:

❖ Lys, and Arg are positively charged amino acids.

❖ Trypsin contains acidic amino acids.

❖ Phe, tyr, and Trp are aromatic amino acids.

❖ Gly, Ala, and Val are small non-polar amino acids.

Features of active site

1. Binding occurs at least three points, because of Chirality (mirror image is not

superimposable).

2. It is a three-dimensional pocket or cleft formed by groups that come from different parts of

the primary structure usually forming a domain.

❖ In other words: Active site is formed of different amino acids. In primary structure,

amino acids were away from each other, but in the tertiary structure, amino acids

are close to each other.

Phe, Tyr, Trp

Lys,

Arg Gly, Ala, Val

3. It is small relative to the total structure of an enzyme. The “extra” amino acids create the

3D active site.

❖ The remaining amino acids may make up regulatory sites.

4. It looks like a canal, cleft, or crevice. It contains nonpolar as well as polar residues. Water

is usually excluded unless it is part of the reaction.

❖ In other word: Active sites are embedded in the enzyme (not on the surface). This is

important to block water (Reactive) from reaching the active site to prevent

reaction interruption and interactions with ant molecule (Specificity).

5. Substrates bind to enzymes by multiple weak attractions.

How do substrates fit into the active site of enzymes?

How do enzymes accelerate reactions?

Lock-and-key model (old)

Here, the substrate fits directly into the active site.

Reasons why this model was rejected:

❖ Proteins are dynamic in nature.

❖ Some enzymes can bind different substrates.

❖ Some enzymes catalyze multi-substrate reactions.

Induced fit model

Enzymes are flexible and active sites can be modified by binding of substrate.

Types of energy

There are two forms of energy:

❖ Potential - capacity to do work (stored).

❖ Kinetic - energy of motion.

Potential energy is more important in the study of biological or chemical systems.

Molecules have their own potential energy stored in the bonds connecting atoms in molecules.

❖ It is known as free energy or G (for Josiah Gibbs).

❖ It is the energy that is available for reactions.

Free energy (G)

The difference between the free energy values between reactants and products (free-energy

change ∆G):

∆G = Gproducts – Greactants

❖ ∆G accounts for the equilibrium of the reaction and enzymes accelerate how quickly this

equilibrium is reached.

***Notes about the above equation:

❖ If G is negative, Gproducts is less than Greactants, energy is not needed to drive the reaction,

but released, making the forward reaction (from left to right) spontaneous (the reaction

is called exergonic).

❖ If G is positive, Gproducts is more than Greactants, an input of energy is needed, making the

reaction not spontaneous (the reaction is called endergonic).

✓ The reverse reaction is exergonic and, thus, spontaneous.

❖ If G is zero, both forward and reverse reactions occur at equal rates; the reaction is at

equilibrium.

***Summary:

∆G < 0 ∆G > 0

Greactants > Gproducts Gproducts > Greactants

G value decreases G value increases

Spontaneous Not Spontaneous

Exergonic Endergonic

Catabolism Anabolism

***Exercise: Draw a line presenting the Ea of the following reaction:

The answer is the red arrow in the second picture.

What do enzymes do?

Any enzymatic reaction goes through a transition state (ES) that has a higher free energy than

does either S or P.

The difference in free energy of the transition state and the substrate is called the activation

energy.

Enzymes lower the activation energy, or, in other words, enzymes facilitate the formation of the

transition state at a lower energy.

At the highest energy level, the substrate configuration is most unstable and is most tightly

bound to the enzyme.

The bonds or the electronic configuration are maximally strained.

Alternative pathways

Substrates often undergo several transformations when associated with the enzyme and each

form has its own free energy value.

The activation energy corresponds to the complex with the highest energy.

The energy of activation does not enter the final ΔG calculation for a reaction.

❖ Example: Adenosine Deaminase.

***Note: Ea (Activation Energy) in multi transition states-reactions can be calculated by:

Ea = TS (Highest one) – SEnergy

How do enzymes catalyze reactions?

Proximity of substrates together.

❖ Make the substrates close to each other and in the right orientation, to increase probability

of collisions (reaction).

Orientation of the active site to fit the substrate in the best fit possible.

Changing the energy within bonds allowing the breakup and formation of bonds.

Catalysis is the end result.

Examples of possible mechanisms to do so:

❖ In some cases, enzymes use a combination of these mechanisms.

Catalysis by bond strain

The induced structural rearrangements produce strained substrate bonds, which more easily

attain the transition state. The new conformation often forces substrate atoms and bulky

catalytic groups into conformations that strain existing substrate bonds.

Example: lysozyme.

The substrate is distorted from the typical 'chair' hexose ring into the 'sofa' conformation, which

is similar in shape to the transition state.

Catalysis involves acid/base reactions

The R groups of amino acids act as donors (acids) or acceptors (bases) of protons.

❖ Examples: histidine, aspartate.

***Exercise: In the stomach, gastric acid decreases the pH to 1 to 2 to denature proteins through

disruption of hydrogen bonding. The protease in the stomach, pepsin, is a member of the

aspartate protease superfamily, enzymes that use two aspartate residues in the active site for

acid–base catalysis of the peptide bond. Why can they not use histidine like chymotrypsin?

❖ Answer: To participate in general acid–base catalysis, the amino acid side chain must be

able to extract a proton at one stage of the reaction and donate it back at another.

Histidine (pKa = 6.0) would be protonated at this low pH and could not extract a proton

from a potential nucleophile. However, aspartic acid, with a pKa of ~2 can release protons

at a pH = 2. The two aspartates work together to activate water through the removal of a

proton to form the hydroxyl nucleophile.

Covalent catalysis

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).

Functional amino acids in active sites

Table 8: Some Functional Groups in the Active Site

FUNCTIONAL OF AMINO ACID ENZYME EXAMPLE

Covalent intermediates

Cysteine−SH Glyceraldehyde 3-phosphate dehydrogenase

Serine−OH Acetylcholinesterase, chymotrypsin

Lysine−NH2 Aldolase

Histidine−NH Phosphoglucomutase

Acid−base catalysis

Histidine−NH Chymotrypsin

Aspartate−COOH Pepsin

Stabilization of anion formed during the reaction

Peptide backbone−NH Chymotrypsin

Arginine−NH Carboxypeptidase A

Serine−OH Alcohol dehydrogenase

***Note: You need to memorize the Functional Groups of Amino Acids.

Naming of enzymes

In general, enzymes end with the suffix (-ase).

Most other enzymes are named for their substrates and for the type of reactions they catalyze,

with the suffix “ase” added.

❖ ATPase breaks down ATP.

❖ ATP synthase synthesizes ATP.

Some enzymes have common names:

❖ Examples: the proteolytic enzyme trypsin & Chymotrypsin.

Exercise: Predict the function of the following enzymes.

1. Maltase: Hydrolysis of maltose.

2. Lactate dehydrogenase: Dehydrogenation of lactate.

Extra (Not required)

Enzyme names give information about their function rather than the structure; focus on type of

reaction catalyzed and the substrate (the reactant). The rules are:

1. The suffix —ase identifies a substance as an enzyme. Examples: Lipase, sucrase.

❖ Some enzymes have the suffix —in. Examples: Trypsin, pepsin.

2. The type of reaction catalyzed is often noted with a prefix. Example: Oxidase catalyzes

oxidation.

3. The substrate is often noted. Examples: Glucose oxidase, pyruvate carboxylase.

❖ When the type of reaction is omitted, it is hydrolysis. Examples: Urease, lactase.

3. Fructose Oxidase: Oxidation of fructose.

4. Maleate isomerase: Isomerization of maleate.

Enzyme Classification – Structure

We classify enzymes according to the structure into:

1. Simple enzymes: Contain only protein, and can be a monomer or a polymer.

2. Complex enzymes: Contain protein part & non-protein part. They are classified into:

❖ Holoenzymes: They are catalytically active enzymes consisting of an apoenzyme

(Protein Portion) combined with its cofactor (Non-protein Portion).

❖ Apoenzymes: a protein that forms an active enzyme system by combination with a

coenzyme.

To summarize it up:

Enzyme Classification – Function

Enzymes are classified into six major groups:

1. Oxidoreductases (EC1).

2. Transferases (EC2).

3. Hydrolases (EC3).

4. Lyases (EC4).

5. Isomerases (EC5).

6. Ligases (EC6).

We can also refer to enzymes by numbers (See above) like: Trypsin is EC 3.4.21.4

❖ 3 refers to the class.

❖ 4 refers to the subclass.

❖ 21 refers to the sub-subclass.

❖ 4 refers to the sub-sub-subclass.

Now let’s discuss each class separately:

Enzymes

Simple Only protein

Complex

Apoenzyme Protein part

Holoenzyme Apoenzyme + Coenzyme

Oxidoreductases (EC1)

They catalyze oxidation/reduction reactions involving the transfer of hydrogen atoms or

electrons.

It is the largest class.

They can be divided into 4 main classes:

1. Dehydrogenases:

Dehydrogenases transfer electrons in the form of hydride ions (H−) or hydrogen atoms using an

electron-transferring coenzyme, such as NAD+ (Oxidized)/NADH (Reduced)or FAD

(Oxidized)/FADH2 (Reduced).

❖ Example 1: Lactate dehydrogenase.

❖ Example 2: Alcohol dehydrogenase.

✓ Notice how the two reactions are reversable.

✓ Notice the coenzymes used in the above reactions.

2. Oxidases:

Oxidases catalyze hydrogen transfer from the substrate to molecular oxygen producing

hydrogen peroxide as a by-product.

Glucose oxidase catalyzes this reaction:

β-D-glucose + O2 gluconolactone + H2O2

3. Peroxidases:

Peroxidases catalyze oxidation of a substrate by hydrogen peroxide.

See the structure of the glutathione.

Example: oxidation of two molecules of glutathione (GSH) in the presence of hydrogen

peroxide:

✓ Notice that Glutathione (Tripeptide) is made of Glutamate, Cysteine, & Glycine.

✓ Methionine is another amino acid containing thiol (Mercpto) group.

✓ Note: We call the amino acid Glutamate instead of Glutamic acid because the

functional group is COO− not COOH.

✓ Glutathione protects our bodies from oxidizing agents by getting oxidized (Losing H

atom form thiol group).

4. Oxygenases:

Oxygenases catalyze substrate oxidation by molecular oxygen through introducing oxygen into

the substrate.

❖ Monooxygenases (AKA: hydroxylases): one oxygen is incorporated into the substrate

and the other produces water (not H2O2).

❖ Dioxygenases: both oxygen atoms are incorporated into the substrate.

Examples: lactate-2-monooxygenase and tryptophan 2,3-dioxygenase.

✓ When we use one Oxygen atom, the enzyme is monooxygenases.

✓ When we use two Oxygen atoms, the enzyme is dioxygenases.

Transferases (EC2)

These enzymes transfer a functional group (C, N, P, or S) from one substrate to an acceptor

molecule. (Transferases make phosphorylation).

❖ Example 1: Kinases (the transferred group is a phosphate)

✓ Phosphofructokinase catalyzes transfer of phosphate from ATP to fructose-6-

phosphate:

✓ Notice the name bisphosphate. We use the term bis when the two phosphate groups are on

two different places.

✓ We use the term di, when the two phosphate groups are on the same place.

❖ Example 2: Transaminases

✓ A transaminase transfers an amino functional group from one amino acid to a keto

acid, converting the amino acid to a keto acid and the keto acid to an amino acid.

✓ Interconversion of certain amino acids.

✓ Aspartate transaminase:

✓ Notice that the reaction is reversible.

✓ The reaction happens as needed.

Hydrolases (EC3)

They catalyze cleavage reactions using water across the bond being broken or the fragment

condensations.

Proteases, esterases, lipases, glycosidases, phosphatases are all examples of hydrolases

named depending on the type of bond cleaved.

Remember (Enzyme Nomenclature): When the type of reaction is omitted from the name of the

enzyme, it is hydrolysis.

❖ Example 1: proteases − A class of hydrolytic enzymes is proteases that catalyze

proteolysis, the hydrolysis of a peptide bond within proteins.

❖ Example 2 - Specific examples: digestive enzymes.

✓ Proteolytic enzymes differ in their degree of substrate specificity.

✓ Trypsin breaks up peptide bonds only on the carboxyl side of Lys and Arg.

✓ Chymotrypsin hydrolyzes peptide bonds involving bulky aromatic amino acids.

✓ Elastase hydrolyzes peptide bonds involving small, uncharged groups such as

Ala, Val, or Gly.

Lyases (EC4)

Lyases cleave C-C, C-O, C-N, and other bonds by elimination, leaving double bonds or rings, or

conversely adding groups to double bonds without hydrolysis (Without using water).

❖ Dehydrases: Removal of H2O from substrate to give double bond. Example: enolase.

❖ Decarboxylases: Replacement of a carboxyl group by a hydrogen. Example: pyruvate

decarboxylase.

❖ Synthases: Addition of a small molecule to a double bond. Example: citrate synthase.

❖ Specific example 1: aldolase − Aldolase breaks down fructose-1,6-bisphosphate into

dihydroxyacetone phosphate and glyceraldehydes-3-phosphate by forming and cleaving

an aldol (hydroxy aldehyde).

❖ Specific example 2: Enolase converts 2-phosphoglycerate to phosphoenolpyruvate by

formation of double bonds.

Isomerases (EC5)

These enzymes catalyze intramolecular rearrangements.

❖ Phosphoglucoisomerase isomerizes glucose-6-phosphate to fructose-6-phosphate:

❖ Posphoglycerate mutase transfers a phosphate group from carbon number 3 to carbon

number 2 of phosphorylated glycerate:

Something different 1: Ribozymes

Most enzymes are proteins, but RNA molecules can act as enzymes, too.

Ribozymes are enzymes made of both protein and RNA.

For some, catalysis is performed by RNA.

❖ Examples include those involved in RNA splicing and protein synthesis in ribosomes.

The catalytic efficiency of RNAs is less than that of protein enzymes but can be enhanced and

stabilized by the presence of protein subunits.

Ligases (EC6)

Ligases join C-C, C-O, C-N, C-S and C-halogen bonds.

The reaction is usually accompanied by the consumption of a high energy compound such as

ATP and other nucleoside triphosphates.

❖ Example 1 (Carboxylases): pyruvate carboxylase:

pyruvate + HCO3- + ATP Oxaloacetate + ADP + Pi

❖ Example 2 (Synthetases): Merging two nucleotides.

Something different 2: Abzymes

Abzymes are antibodies (immunoglobulin) acting as enzymes.

❖ Characteristics of Abzymes:

✓ High affinity & very specific.

✓ Can convert molecules into products.

✓ Used for treating heroin addicted people.

They are produced against transition-state

analogs.

How? A host animal is injected with a transition-

state analogue. The animal makes antibodies

against it (binding with high affinity at specific

binding points mimicking an enzyme’s active site

surrounding a transition state.

To Summarize it up:

Main Class Subclass Type of reaction catalyzed

Oxidoreductases (EC1)

Dehydrogenases Introduction of double bond (oxidation) by formal removal of two H atoms from substrate, the H being accepted by a coenzyme

Oxidases Oxidation of a substrate

Peroxidases Oxidation of a substrate by hydrogen peroxide

Oxygenases Substrate oxidation by molecular oxygen

Transferases (EC2) Transaminases Transfer of an amino group between substrates

Kinases Transfer of a phosphate group between substrates

Hydrolases (EC3)

Proteases Hydrolysis of amide linkages in proteins (Peptide bonds)

Esterases Hydrolysis of ester linkages

Lipases Hydrolysis of complex ester linkages in lipids

Glycosidases Hydrolysis of glycosidic bonds in carbohydrates

phosphatases Hydrolysis of phosphate-ester bonds

Lyases (EC4)

Dehydratases removal of H2O from a substrate

Decarboxylases removal of CO2 from a substrate

Synthases Addition of a small molecule to a double bond

Isomerases (EC5) Isomerases Changing from one isomer to another

Mutases transfer of a functional group from one position to another in the same molecule

Ligases (EC6) Carboxylases

Formation of new bond between a substrate and CO2, with participation of ATP

Synthetases Formation of new bond between two substrates, with participation of ATP

The End

Do not forget to answer the test bank.