Introduction to Biochemistry (02215852)

Amino acids and Proteins

Function Amino acids: the building blocks
- Catalysts
- Transport/
- store molecules (e.g.O2)
- Mechanical support
- Immune protection
- Generate movement
- Transmit nerve impulses
- Control growth and
differentiation.

Properties of Amino acids

- Size and shape, Charge
⚫ Protein is a nitrogenous substance found in the protoplasm of all animal
- Polarity, Hydrophobicity
and plant cells.
- Aromaticity
⚫ About twenty percent of the human body is composed of proteins.
- Conformation – usually by side
⚫ Proteins are large, complex molecules crucial for the normal functioning of
chain
cells.
- Propensity to adopt a particular
⚫ Proteins are comprised of smaller units called amino acids, which are the
conformation
building blocks of proteins, linked together by peptide bonds to form long
- Relative position in protein
chains.

It contains mainly carbon, hydrogen, oxygen, nitrogen, and sulfur atoms in its
structure. Other elements such as phosphorus or iron are present in nucleoprotein
and hemoglobin.

Proteins are natural polymers of amino acids.

The number of amino acids in a protein molecule may range from two to
several thousands

Protein molecules contain Nitrogen, Carbon, Hydrogen, and Oxygen

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Special amino acids
1. Proline's uniqueness lies in its structural feature where its side chain is
connected to the protein backbone twice, forming a five-membered
nitrogen-containing ring.
2. It is the sole proteinogenic secondary amino acid with a secondary amine
configuration, owing to its nitrogen atom being attached both to the α-
carbon and to a chain of three.
Special amino acids: Cysteine and cystine
1. Cysteine possesses a sulfur group located at the end of its molecular
structure.
2. Adjacent cysteine residues in a protein's structure can spontaneously form
a covalent bond called a disulfide bond under specific conditions.
3. This bond formation results in the creation of a compound called cystine.
Amino acid is a Chirol Molecule
⚫ Chirality: Property of molecules having two non-superimposable mirror-
image forms (enantiomers).
- Chiral center (chiral carbon) is a carbon atom that has four different groups
attached to it.
⚫ Enantiomers: Mirror-image forms that rotate polarized light differently; right-
handed to the right, left-handed to the left.
⚫ Amino acids: Chiral molecules crucial for protein synthesis.
⚫ Homochirality: All amino acids in organisms are exclusively left-handed,
demonstrating a universal preference in nature.
D and L configuration
[D and L configuration refer to the absolute configuration of a molecule based on the
orientation of its chiral Centre(s) relative to glyceraldehyde]
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Question1(***Final Exam Open Question↑***)

Between 20 amino acids, 19 of them are chirol.

Why organisms only use left-handed amino acids to make protein
The amino acid Glycine has hydrogen as the R group.
Answer:
There is no priority between them
In living organisms, only left–handed amino acids are used to make proteins.

◼ Enzyme specificity: Catalyzing peptide bond formation, enzymes

exclusively recognize and link left-handed amino acids.
Primary structure: Trans and cis peptide bonds
◼ Exclusion of right-handed amino acids: Despite their presence, enzymes
do not incorporate right-handed amino acids into proteins.
◼ Biological preference: Organisms use only left-handed amino acids for
protein synthesis due to enzyme specificity.
R – S configuration to Chirol Center’
To assigh the R – S configuration we need
to rank these four groups using the con –
angle pre-log process.
Group 1 has the highest priority based on
atomic number.
⚫ Trans and cis peptide bonds: Two configurations of peptide bonds.
⚫ Almost all peptide bonds are trans: Trans configuration is predominant in
proteins.
⚫ Steric hindrance: Occurs in the cis configuration due to repulsive forces
between functional groups.
⚫ Steric effects: Result from overlapping electron clouds, preventing
molecules from being in close proximity.
⚫ Trans configuration preferable: Due to reduced steric hindrance, trans
configuration is favored over cis configuration in proteins.

R and S are enantiomer of each others.

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⚫ Essential amino acids (EAAs): These must be obtained from the diet as Ka : Acid Dissociation Constant Pka( *** Very Important***)
the body cannot synthesize them sufficiently.
⚫ Plant proteins may lack specific EAAs, but combining sources like cereals
and legumes can provide a complete set.
⚫ Some plants like quinoa contain better amounts of essential amino acids,
enhancing dietary variety and balance.

Amino acids are Amphoteric

◼ pKa = -logKa
- All amino acids are amphoteric compounds
◼ pKa of acetic acid is 4.76 → it is not dissociated in water
⚫ Structure dependence on pH: Amino acids contain carboxyl (-COOH) and
completely → a weak acid
amino (-NH2) groups, which can donate or accept protons, affecting their
◼ pKa of HCl is -8.3 → It is dissociated in water completely → A
overall charge and structure based on pH.
strong acid
⚫ Amphoteric property: Amino acids exhibit an amphoteric nature due to
➢ Henderson-Hasselbalch equation: Links pH, pKa, and the ratio of a
possessing both acidic and basic functional groups, allowing them to act
conjugate base to its acid form.
as both acids and bases depending on the pH of their surroundings.
➢ Ka: Measures acid strength by quantifying its tendency to dissociate.
➢ Strong acids have high Ka values, indicating greater dissociation.
➢ Kb: Describes base ionization into positive and negative components.
➢ pKa: pH at which 50% of a substance is ionized, reflecting its acidity or
basicity.

The R group determines the hydrophobicity, size, charge, secondary structure

𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒
preference, and Aromaticity pH = pKa + 𝑙𝑜𝑔 (for weak acid)
𝑤𝑒𝑎𝑘 𝑎𝑐𝑖𝑑

𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑎𝑐𝑖𝑑
pOH = pKb + 𝑙𝑜𝑔 (for weak base)
𝑤𝑒𝑎𝑘 𝑏𝑎𝑠𝑒
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pKa in amino acids pka of the carboxylic group is 2-3=>

➢ pKa determines the protonation state and charges of amino acids' when pH< 2-3, it is protonated=>COOH
functional groups.
When pH> 2-3, it is deprotonated=COO-
➢ Amino acids possess at least two functional groups: amino (-NH2) and
carboxyl (-COOH), which can behave as either acids or bases.
➢ The pKa of these functional groups dictates the pH at which they become
protonated or deprotonated, impacting the overall charge of the amino
acid molecule.

At physiological pH (7.4)

For amino group: pH protonated form=> NH3+

For carboxylic group:pH>pKa=> deprotonated form=> COO-

(***Final Exam***)

pka of the amino group is 9-10=>

when pH< 9-10, it is protonated=> NH3+

When pH>NH2, it is deprotonated=> NH2

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Amino acids: Ionization state

- Amino acids in water form zwitterions, acting as both acids (proton

donors) and bases (proton acceptors).

(*** Protonate ***)

Titration of Glycine

Isoelectric point (pI)

➢ Isoelectric point (pI): pH where a molecule is electrically neutral.

➢ Calculated using Henderson-Hasselbalch equation: pI = 1/2 (pK1 + pK2).
➢ For amino acids, pI is the average of pKa values of ionizable groups with
positive and negative charges.
➢ Example: Glycine's pI ≈ 6.0, averaging carboxyl and amino group pKa
values. ➢ Most amino acids resemble Gly in ionization properties, but 5 exceptions
➢ Side chain impact: Amino acids with ionizable side chains, like histidine, (Glu, Asp, Lys, Arg, His) have polar charged side chains.
consider side chain pKa along with carboxyl and amino groups for pI
Positive charge  PI →negative charge
calculation.
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The isoelectric point of amino acids pka values of amino acids with Ionizable R-group

pI is the pH that charges on amino acid is zero.

( 항 PI를 기준으로, negative, positive charge로 기준나누기)

We are mostly
concerned with these 5
Amino acid residues.
Because their effect is
more prominent in their
presence in proteins

pKaR (R group) renders the form of titration curve and causes new properties

for amino acids.

pI for Aspartic and Glutamic (acidic amino acids)

Pk1 < pKR < pk2

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pI for Lysine, Arginine, and Histidine (basic amino acids) PI for dipeptides

1. Draw the peptide at its most protonated form (low pH).

2. Calculate the overall charge

3. calculate the change in charge as pH rises.

4. Use 2 pkassurrounded peptide at Zero charge

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UV spectrophotometry Proteins: composition, structure, and function

⚫ UV spectrophotometry: Analyzes absorption/transmission of UV/visible Proteins:
light by a sample
◼ Definition: Molecules yielding amino acids upon hydrolysis
⚫ Components: Light source, monochromator, sample container, detector,
◼ Functions: Structural components, enzymes (biological catalysts),
signal processor
immunity molecules, fuel providers
⚫ Provides insights into sample composition and concentration
◼ Varied amino acid count: Ranging from two to several thousand
Chromophores present in proteins ◼ Elemental composition: Nitrogen, Carbon, Hydrogen, Oxygen

- Chromophores: A chemical group that absorbs light at a specific frequency

and so imparts color to a molecule

UV-visible absorbance:

◼ Near UV region: 250-500 nm

◼ Far UV region: 190-250 nm
◼ Chromophoric parts: Trp, Tyr, Phe (near UV, max absorption at 280 nm),
Disulfide bonds (near UV, absorbance near 260 nm)
◼ Peptide bond: Major chromophore in far UV (strong absorption at 190 nm,
weak bands at 210-220 nm)
◼ Oxygen: Absorption measured at 205 nm
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Classification of Proteins Conjugated Proteins:

Definition: Contain non-protein prosthetic group attached to protein part

Types: Nucleoproteins, Chromoproteins, Glycoproteins, Phosphoproteins,

Lipoproteins, Metalloproteins

Derived Proteins:

Types: Primary and Secondary

Primary Proteins: Insoluble in water, soluble in acids and alkalis, slight changes in
properties, little hydrolytic cleavage

Secondary Proteins: Soluble in water, coagulated by heat, formed by progressive

hydrolytic cleavage, includes proteoses, peptones, peptides

Primary Protein Derivatives:

Proteins: Insoluble products from water, dilute acids, enzymes, e.g., myosan, fibrin

Metaproteins: Formed by acids/alkalies, insoluble in neutral solvents

Coagulated Proteins: Insoluble products from heat/alcohol, e.g., cooked meat,

cooked albumin
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Protein synthesis: “ Translation” Protein Primary Structure:

◼ Definition: Linear sequence of amino acids, connected by covalent bonds

◼ Peptide Bond: Formed by linking the α-carboxyl group of one amino acid
to the α-amino group of another
◼ Condensation Reaction: Also known as dehydration synthesis
◼ Restricted Rotation: Peptide bond behaves like a partial double bond due
to limited rotation

Peptides' Directionality:

◼ Peptides Have Direction: The sequence of amino acids in a peptide chain

progresses from the N-terminus to the C-terminus

<Primary structure>

Structures of Proteins (Four levels of structure)

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<Peptide bind> Characteristics:

◼ Hydrogen Bonding: Occurs between NH and CO groups, except at helix

termini.
◼ Dense Structure: Helix core is tightly packed with no internal space.
◼ Right-Handed Twist: Helix has a right-handed spiral.
◼ Proline Effect: Proline disrupts helix formation due to lack of NH group.
◼ R-Group Orientation: R-groups project outward from the helix.

Pitch: Distance along the helix axis for one complete turn.

Rise: Distance between two consecutive amino acids along the helix axis.

<Peptides have direction>

Parallel and Anti-Parallel Beta-Sheet:

◼ Structure: Sheet-like arrangement of polypeptide strands running either

parallel or anti-parallel to each other.

Characteristics:

◼ Hydrogen Bonding: Occurs between NH and CO groups of adjacent

strands.
◼ Parallel: Strands run in the same direction, resulting in longer, weaker
hydrogen bonds.
◼ Anti-Parallel: Strands run in opposite directions, leading to shorter,
Secondary Structure: stronger hydrogen bonds.
◼ Stability: Anti-parallel sheets are generally more stable than parallel sheets.
Alpha-Helix:

◼ Structure: Helical shape formed by hydrogen bonding between NH and

CO groups.
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Secondary Structure

Q. Why B – pleated sheet?

Structure:

◼ Not Flat: Beta-sheets are not flat but pleated due to the tetrahedral nature
of the α-carbon.
◼ Tetrahedral Carbon: The α-carbons in the polypeptide backbone are
tetrahedral, introducing a kink in the sheet.
◼ Connected Planes: Despite having planes, they are connected by
Parallel and Anti-Parallel Beta-Sheet tetrahedral carbons, causing a slight kink in the sheet.

R-Group Orientation:

◼ Above or Below Plane: R-groups of amino acids in the sheet are oriented
either above or below the plane of the sheet.
◼ Alternate Orientation: Adjacent R-groups alternate in orientation; if one R-
group is below the plane, the next is above it.

Answer: Beta-pleated sheets are named for their pleated or folded

structure, resulting from the tetrahedral nature of α-carbons in the
polypeptide backbone. This tetrahedral arrangement introduces kinks,
causing the connected planes of the sheet to fold, creating a
characteristic pleated appearance.
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Secondary Structure Tertiary Structure

[Beta-turn or reverse Turns] Definition: Overall three-dimensional arrangement of a protein's atoms.

Protein Structure: Key Points:

◼ Compact, Globular Shapes: Common for most proteins. The global folded pattern in 3D.
◼ Owing to Polypeptide Chain Reversals: Reversals in direction contribute to
➢ Includes longer-range aspects beyond secondary structure.
compactness.
➢ Influenced by specific folding patterns unique to each protein.
Reverse Turn (Hairpin Turn):

◼ Common Structural Element: Facilitates reversals in polypeptide chain

direction.
◼ Also Known As Turn or hairpin turn.

One way for strands to change direction.

There are 6 types of B-turn structure that
all have hydrogen bonds between the
carbonyl of residue i and the N-H group of
residue i+3.

Loops in Protein Structure:

◼ Extended forms of turns, often termed Ω loops, are found on protein

surfaces.
◼ These regions play vital roles in protein-protein and protein-ligand
interactions.
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Stabilization:: Interacting segments of polypeptide chains are held in characteristic Orientation in Water:
positions by:
➢ Hydrophilic Groups: Oriented towards the water interface.
➢ Non-covalent interactions: Including hydrogen bonds, van der Waals ➢ Hydrophobic Groups: Oriented away from the water interface.
forces, and electrostatic interactions.
➢ Disulfide bonds: Covalent cross-links between segments, enhancing
stability.

Quaternary Structure:

Tertiary protein structure=complete folding pattern(Sickle cell disease)
Specific Folding: Not random, unique to each protein, essential for its function.
Factors Affecting Folding:
➢ Hydrophobic Residues: Typically located in protein interior to avoid contact
with aqueous solvent.
➢ Hydrophilic Residues: Found on protein surface to interact with water,
maximizing electrostatic interactions.
Stabilization Mechanisms:
➢ Definition: Relationship of two or more polypeptide chains forming a
complex.
➢ Composition: Proteins may contain multiple separate polypeptide chains
(subunits), identical or different.
➢ Arrangement: Three-dimensional arrangement of protein subunits
constitutes quaternary structure.
Bonding in Polypeptide Assembly:
➢ Hydrogen Bonds: Weak interactions between polypeptide chains.
➢ Covalent Bonds: Disulfide bonds between cysteine residues.

➢ Disulfide Bonds: Formed between cysteine residues, enhancing structural

integrity through covalent linkages.
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Function of Hemoglobin:

➢ Role: Binds to oxygen for transport in blood, residing in red blood cells.
➢ Structure: Consists of multiple subunits, each containing a heme group
that binds oxygen.
The major protein types

Examples: Fibrous Proteins:

➢ Alpha-Helical Protein: Characterized by numerous helices.
➢ Porin: Transmembrane protein allowing passive transport of hydrophilic
molecules.
Porin Structure:
➢ Hydrophobic Exterior: The outer layer interacts with the hydrophobic
membrane.
➢ Hydrophilic Interior: Forms water channel for molecule transport.
Structure: Long and spindly, composed of elongated, fibrous polypeptide chains.
Composition:
➢ Primary and secondary structures are predominant.
➢ Little to no tertiary structure.
➢ Long parallel polypeptide chains with cross-linkages between them.
Properties:
➢ Mostly insoluble due to non-polar groups on the exterior.
➢ Repetitive amino acid sequence.
➢ Structural roles, less sensitive to temperature and pH.
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Examples: Keratin (in hair and skin), Collagen (in connective tissue), Silk. Conformational Stability:
Globular Proteins: Definition: Refers to the ability of a protein to maintain its folded 3D structure, which

Structure: Highly folded and compact, forming a spherical/globular shape. is essential for its activity.

Composition: Contributing Forces:

➢ Primary and secondary structures are present, along with complex tertiary ➢ Peptide Bonds: Provide stability in the primary structure.

structures. ➢ Hydrogen Bonds: Backbone interactions crucial for secondary structure

➢ Some may have a quaternary structure. stability.

➢ Non-polar groups are typically inside, polar groups outside. ➢ Tertiary Structure Interactions: Distant interactions within a single protein,
including Van der Waals forces, hydrophobic interactions, and disulfide
Properties: bonding, in addition to hydrogen bonds.

➢ Usually soluble in water. ➢ Quaternary Structure Interactions: Interactions between individual protein

➢ Irregular amino acid sequence. subunits, determined by similar bonds as those in tertiary structure.

➢ More sensitive to temperature and pH changes. Functional Importance:

Examples: Enzymes, antibodies, hemoglobin. ➢ Proteins are only functional when they are in their proper conformation,
highlighting the significance of conformational stability for protein function.
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[Note]
Give the name of the following 5 amino acids
A; Lysin, B: Leucine, C: Proline, D: Threonine, E: Tyrosin
Denaturation of Protein:
Definition: Biochemical process involving alterations in secondary, tertiary, or
quaternary protein structures, leading to disruption of covalent bonds.
Process: Protein loses its native shape due to disruption of weak chemical bonds
and interactions, rendering it biologically inactive.
Factors Affecting Denaturation:
➢ Temperature and pH fluctuations.
➢ Disruption of weak chemical bonds and interactions.
Effects: Denatured protein becomes improperly folded or degraded, resulting in loss
of biological activity.
Solvation Shell:
➢ Definition: Layer of solvent surrounding a protein, contributing to its
stability.
➢ Role: Helps stabilize protein conformation by interacting with charged
amino acid residues on the protein's exterior.
Importance of Conformational Stability:
➢ Crucial: The stability of protein conformation depends not only on
interactions contributing to primary, secondary, tertiary, and quaternary
structures but also on the surrounding environment.
➢ Significance: Proper folding of proteins is essential for maintaining their
functionality.
Q. What happens if things go wrong?
Denaturation Factors:
➢ Temperature: Changes cause proteins to lose their shape and function.
➢ pH: Alterations disrupt ionic bonds, affecting tertiary and quaternary
structure.
➢ Chemical Denaturants: Disrupt hydrogen bonding within proteins.
Effects on Protein Structure:
➢ Hydrogen Bonds: Disrupted by chemical denaturants, impacting
secondary, tertiary, and quaternary structure.
➢ Primary Structure: Generally preserved despite changes in temperature,
pH, or chemical environment.
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Impact of Charges: Organic Compounds (Alcohol, Urea, Guanidinium Hydrochloride):

➢ Disruption of Balance: The addition of positive or negative charges ➢ Effects: Disrupt non-covalent interactions between amino acids.
disturbs interactions between charged amino acids, affecting protein ➢ Examples: Ethanol and isopropyl alcohol in wound disinfection.
stability.
Ions (Heavy Metals):

➢ Effects: High salt concentrations disrupt electrostatic interactions.

➢ Examples: Mercury and lead poisoning.

Agitation (Mechanical Denaturation):

➢ Effects: Mechanical force disrupts non-covalent interactions.

➢ Examples: Whipped cream, and meringue preparation.

Overall, these agents disrupt protein structure and function by interfering with
various bonds and interactions within the protein molecules, leading to denaturation.

[Denaturing Agents and Effects]:

Heat (Above 50°C) Changes in Denatured Proteins:

➢ Bonds Disrupted: Hydrogen bonds, hydrophobic interactions, van der
Waals forces.
➢ Examples: Cooking food, autoclaving surgical items.
Acids and Bases:
➢ Effects: Altering pH disrupts electrostatic interactions between amino acids.
➢ Examples: Lactic acid in yogurt and cheese preparation.
- Reduced Solubility: Often become insoluble due to unfolding.
- Decrease in Biological Activity: Loss of functional properties.
- Depletion of Crystallizing Property: Inability to form crystals.
- Increased Constituent Group Reactivity: Enhanced susceptibility to
chemical reactions.
- Alteration in Shape: Protein molecules lose their native conformation.
- Susceptibility to Enzymatic Hydrolysis: Easier breakdown by enzymes
due to exposed peptide bonds.
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Strange Proteins Discovery Enzymes Overview:

- Researchers at the University of Tokyo identified a new group of proteins

- Enzymes are biological catalysts that accelerate biochemical
with flexible, string-like structures.
reactions.
- These proteins, dubbed "hero proteins," exhibit unusual shapes and
- Typically, enzymes are three-dimensional globular proteins, possessing
possess heat-resistant properties.
tertiary and quaternary structures.
- They have been found in various animals, from insects to humans, and

show potential in protecting against protein clumps associated with

neurodegenerative diseases.:

Hydrolysis of Proteins:

- Peptide bonds are broken through hydrolysis reactions.

- Enzymes catalyze selective hydrolysis, breaking peptide bonds into amino

acids.
Structure of Enzymes:
- Occurs in the stomach during digestion, facilitated by enzymes known as
- Enzymes consist of an active site, where substrates bind and catalytic
proteases.
reactions occur.
- Factors such as temperature, pH, and enzymatic activity influence the
- Active sites have a specific shape determined by the enzyme's tertiary
process of breaking down protein molecules into smaller peptides and
structure.
amino acids.
- Any alteration in the protein's shape affects the active site and

consequently, the enzyme's function.

- Enzymes exhibit high substrate specificity, recognizing and binding to

particular substrates.
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- Its EC number indicates its classification and function in the glycolysis

pathway.

Enzyme Classification Overview:

- Enzymes are categorized into six main classes by the International Enzyme

Commission (EC).

- Each class is based on the type of reaction catalyzed.

Active Site Components:
EC1. Oxidoreductase:
- The active site is divided into the binding site, which selects and binds
◼ Catalyzes redox reactions.
substrates, and the catalytic site, where the enzyme's catalytic action
◼ Examples: Oxidases, Dehydrogenases, Oxygenases, Peroxidases,
occurs.
Catalases.
Naming and Classification:
◼ Transfer of hydrogen, oxygen atoms, or electrons between substrates.

- Enzymes are often named with names ending in "ase," reflecting their

function.

- The International Enzyme Commission (EC) classifies enzymes based on

the types of reactions they catalyze.

- The EC number system categorizes enzymes with four digits, indicating the

reaction type, substrate, function, and specificity.

Example: Lactase:

- Lactase, which breaks down lactose, is named based on its substrate.

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EC2. Transferases: EC4. Lyases:

◼ Catalyzes the transfer of functional groups between substrates. ◼ Cleaves covalent bonds without water or oxidation.

◼ Examples: Kinases, Transaminase. ◼ Example: Decarboxylases.

◼ Kinases transfer phosphate groups from ATP. ◼ Pyruvate decarboxylase catalyzes the conversion of pyruvate to

◼ Transaminase transfers amino groups between amino acids. acetaldehyde without water or oxidation.

EC5. Isomerases:

EC3. Hydrolases: ◼ Catalyzes intramolecular changes in substrates.

◼ Catalyzes hydrolysis or breakdown reactions. ◼ Example: Glucose isomerase.

◼ Examples: Esterases, Lipases, Proteases. ◼ Converts glucose into its isomeric form.

◼ Hydrolyzes ester, peptide bonds, etc.

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EC6. Synthetases:

◼ Catalyzes the formation of new bonds between molecules.

◼ Joins two molecules to form a new compound.