biochemistry lecture (proteins)

BC 1008 - Structure and

Function of Biomolecules

Devaka Weerakoon (18 L)

and

Dilrukshi de Silva (12 L)

Department of Zoology

(3 Cr – 30L + 30P)

Objectives and Learning Outcomes

• To Introduce the four basic biomolecules, their structure

and function

• What an amino acids is and their properties

• Structure of a protein

• Few examples of fibrous and globular proteins

• What an enzyme is and their functioning

• Structure of Nucleic acids

• Information storage and expression

• What a carbohydrate is and diferent types of carbohydrates

and functions

• What a lipid is and different types of lipids and their functions

Substances Found in Living Organisms

Water

Macromolecules: Giant Polymers

• There are four major types of biological macromolecules:

• Proteins

• Carbohydrates

• Lipids

• Nucleic acids

• These macromolecules are made the same way in all living things, and are present in all organisms in roughly the same proportions

• An advantage of this biochemical unity is that organisms can use these molecules interchangebly

• Macromolecules are giant polymers

• Polymers are formed by covalent linkages of smaller units called monomers

• Molecules with molecular weights greater than 1,000 daltons (atomic mass units) are usually classified as macromolecules


• The functions of macromolecules are related to

the shape and the chemical properties of their

monomers

• Some of the roles of macromolecules include:

• Energy storage

• Structural support

• Transport

• Protection and defense

• Regulation of metabolic activities

• Means for movement, growth, and development

• Information storage


• Macromolecules are made from smaller monomers

by means of a condensation or dehydration

reaction in which an OH from one monomer is

linked to an H from another monomer

• Energy must be added to make or break a polymer

• The reverse reaction, in which polymers are

broken back into monomers, is a called a

hydrolysis reaction

Condensation and Hydrolysis Reactions

Condensation and Hydrolysis of Polymers

How are organic molecules synthesized?

Molecules can be metabolized (broken down)

Condensation and Hydrolysis Reactions

1. Introduction to proteins; Protein structure; fibrous

proteins; myoglobin and haemoglobin; immunoglobulins;

Introduction to enzymes; enzyme kinetics and inhibition;

modes of enzyme catalysis; serine proteases

2.Introduction to nucleic acids; Structure of DNA and RNA;

information storage and retrieval; the genetic code

3.Introduction to lipids; steroids and eicosanoids;

phospholipids and membranes; transport across membranes

4.Introduction to carbohydrates; linear and cyclic

structures; stereochemistry and Fischer projections;

Haworth projections; glycosidic bonds; disaccharides;

polysaccharides and complex carbohydrates

Structure and Function of Biomolecules

Proteins: Polymers of Amino Acids

• Proteins are polymers of amino acids. They are molecules with diverse structures and functions

• Each different type of protein has a characteristic amino acid composition and order

• Proteins range in size from a few amino acids to thousands of them

• Folding is crucial to the function of a protein and is influenced largely by the sequence of amino acids

Protein Functions

1. Structural e.g. Collagen, elastin

2. Mobility e.g. Actin/myosin, tubulin, flagella

3. Receptors e.g. Insulin receptor

4. Ligands e.g. Insulin

5. Defense e.g. Antibodies

6. Housekeeping e.g. Enzymes of glycolysis

7. Signalling e.g. Signalling molecules

8. Enzymes e.g. Proteases

9. Storage e.g. Ovalbumin, casein

10.Transport e.g. Haemoglobin

The Monomeric Unit is the Amino Acids

• An amino acid has four groups attached to a central carbon atom:

• Central carbon atom - a carbon

• A hydrogen atom

• An amino group (NH2)

• A carboxylic group

(COOH)

• Differences in amino acids come from the side chains, or the R group

• Twenty amino acids used by the living organisms for synthesis of proteins

• Amino acids can be classified based on the

characteristics of their R groups

A. Nonpolar hydrophobic side chains

B. Polar but uncharged side chains

C. Charged hydrophilic side chains

D. Special amino acids


Non Polar Hydrophobic R groups

Charged R groups

Polar but Uncharged R groups

Unusual Amino Acids

• Cysteine has a terminal sulphydral group (SH)

• Glycine has a H atom as the side chain

• Proline - the R group forms a covalent bond with

the amino group, forming a ring (imino acid)

Two cysteines can form a Cystine

Amino Acids Display Stereoisomerism

• An isomer is a compound that has the same

molecular formula but exist in different forms

Amino Acids Display Steroeisomerism

• Compounds that carry asymmetric carbon atoms or chiral

centers show optical isomerism i.e. they can cause plane

polarized light to rotate in left or right direction

• Amino acids show stereoisomerism as all of them except

Glycine carry chiral centers

• Amino acids that exist in nature are the L forms

• Hydroxyproline

• Ornithine

• Citrulline

• Thyroxine

Special Amino Acids

• The relationship between the chemical species and dissociation constant is expressed by the Henderson-Hasselbalch equation

pH = pKa + log[A-]/[HA]

Amino Acids can act as Week Acids/ Bases

CH3COOH CH3COO- + H+

Amino Acids can act as Buffers

Amino acids contain a basic amino group and an acidic carboxyl group

Formation of Peptide Linkages

• Proteins are synthesized by a condensation reactions between the amino group of one amino acid and the carboxyl group of another

• This forms a peptide linkage

• Peptide bond has partial double bond character

• Causes linkage to be planar – no rotation around peptide bond

Amino acid linkage results in a Peptide

• Dipeptide – peptide consisting of two amino acids

• Tripeptide - peptide consisting of three amino acids

• Oligopeptide - peptide consisting of several amino

acids

• Polypeptide - peptide consisting of many amino acids

• Some examples of naturally occurring peptides

• glutathione – tripeptide (glu-cys-gly) - scavenger of

free radicals

• leucine enkephalen - naturally occurring analgesic

• Oxytocin – Hormone comprising of nine amino acids

• L-aspartyl – L-phenylalanine - aspartame

The Four Levels of Protein Structure: Primary Structure

• There are four levels of protein structure: primary, secondary, tertiary, and quaternary

• The precise sequence of amino acids is called its primary structure

• The peptide backbone consists of repeating units of atoms: N—C—C—N—C—C

The Four Levels of Protein Structure: Secondary Structure

• A protein’s secondary structure consists of regular, repeated patterns in different regions in a polypeptide chain

• This shape is influenced primarily by hydrogen bonds arising from the amino acid sequence (the primary structure)

• The two common secondary structures are the alpha helixand the beta pleated sheet

• The alpha helix is a right-handed coil

• The peptide backbone takes on a helical shape due to hydrogen bonds.

• The R groups point away from the peptide backbone and stabilize the structure by forming H bonds

• Fibrous structural proteins have a-helical secondary structures, such as the keratins found in hair, feathers, and hooves


• b pleated sheets form from peptide regions that lie parallel to each other

• Sometimes the parallel regions are in the same peptide, sometimes they are from different peptide strands

• This sheet like structure is stabilized by H bonds between N-H groups on one chain with the C=O group on the other

• Spider silk is made of b pleated sheets from separate peptides


Secondary Structure of Proteins

• Other elements of secondary structure include beta turns and omega loops

• Tertiary structure is the three-dimensional shape of the completed polypeptide

The Four Levels of Protein Structure: Tertiary Structure

• The primary determinant of the tertiary structure is the interaction between R groups

• Factors determining tertiary structure:

• The nature and location of secondary structures

• Hydrophobic side-chain aggregation and van der Waals forces, which help stabilize them

• The ionic interactions between the positive and negative charges and hydrogen bonding between polar residues

• Disulfide bridges, which form between cysteine residues

The Four Levels of Protein Structure: Tertiary Structure

• Quaternary structureresults from the ways in which multiple polypeptide subunits bind together and interact

• This level of structure adds to the three-dimensional shape of the finished protein

• Hemoglobin is an example of such a protein; it has four subunits

The Four Levels of Protein Structure: Quatenary Structure

The Four Levels of Protein Structure: Summary

Bonds Contributing to the Structure of a Protein

Irregular

contortions

from bondings

between side

chains.

Hydrophobic

clusters at the

core of

proteins

Primary Structure

• Peptide bond (Covalent)

Secondary, Tertiary and Quaternary Structures

Noncovalent Linkages

Hydrogen 4-20

van der Waals

Hydrophobic

Ionic

Covalent Linkages

Disulphide Bridges

• Chaperonins are specialized proteins that help keep other proteins from interacting inappropriately with one another

• When a protein fails to fold correctly, serious complications can occur

• Incorrectly folded proteins are digested by proteosomes and the amino acids are recycled

Proteins: Chaperon Proteins

• Shape or conformation is crucial to the functioning of proteins

• The final conformation will be governed by the type of amino acids that make up the protein which will influence the folding pattern

• Changes in amino acids can take place due to changes in DNA a process called mutation that can drastically change protein structure and therefore the function


• Changes in temperature, pH, urea, salt concentrations, and oxidation or reduction conditions can change the shape of proteins.

• This loss of a protein’s normal three-dimensional structure is called denaturation.

Protein Denaturation

• In some proteins further modification is needed for functioning

• Glycosylation – adding carbohydrate moieties which takes place in the golgi complex

• Adding lipid moieties especially in membrane proteins

Protein Modification

Membrane Proteins

• Lipid anchored proteins

(a) Glycolipid covalent attachment by glycophosphatidylinositol (GPI anchored proteins)

(b) Covalent attachment of the protein to fatty acid like myristic acid or palmitic acid or the prenyl group (15-C franesyl hydrocarbons with repeating vinyl groups)

• In some proteins further modification is needed for functioning

• Glycosylation – adding carbohydrate moieties which takes place in the golgi complex

• Adding lipid moieties especially in membrane proteins

• Covalent modification e.g. acetylation and methylation of Lys, methylation of Arg and His, phosphorylation of Ser, Thr or Tyr

• Sometimes they need prosthetic groups

• Sometimes cleavage is necessary for final action

Protein Modification

Domains

• The term domain is used to describe an area of a protein which is functionally or physically distinct

• Steroid Hormone Receptors

• Another example would be transmembrane proteins that have cytosolic, transmembrane and extracellular domains

DNA binding domain

Hormone binding domain

Transcription activating domain

Inhibitory protein complex

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Globular and Fibrous Proteins

Globular proteins Fibrous proteins

“spherical” shape long, thin fibers

Insulin Hair

Hemoglobin Wool

Enzymes Skin

Antibodies Nails

Fibrous proteins

• Proteins which are folded to a more or less rod like shape

• They

• consist of long fibers or large sheets

• tend to be mechanically strong

• are insoluble in water and dilute salt solutions

• play important structural roles in nature

• Involved in structure: tendons ligaments blood clots, hair, hooves feathers etc., (e.g. Collagen, elastin, keratin and fibrin)

Fibrous Proteins

Keratin:

• Long, fiber-like shapes

• Typically structural

• Ex: a-keratins

hair, wool, skin, and nails

3 a-helices held together by disulfide bonds

• Ex: b-keratins

Feathers, scales

large amounts of beta-pleated sheet structure

• Collagen

• Connective tissue, skin, tendons, and cartilage

• Consists of three polypeptide chains wrapped around each other in a ropelike twist to form a triple helix called tropocollagen; MW approx. 300,000

• 30% of amino acids in each chain are Pro and Hyp

(hydroxyproline); hydroxylysine also occurs that

contain –OH groups for hydrogen bonding

Fibrous Proteins

Collagen Triple Helix

• Every third position is Gly and repeating sequences are X-Pro-Gly or X-Hyp-Gly

• The three strands are held together by hydrogen bonding involving hydroxyproline and hydroxylysine

• With age, collagen helices become cross linked by covalent bonds formed between Lys and His residues

• Deficiency of Hyp results in fragile collagen

Globular Proteins

• Proteins which are folded to a more or less spherical shape

• They

• Tend to be soluble in water and salt solutions

• Most of their polar side chains are on the outside and interact with the aqueous environment by hydrogen bonding and ion-dipole interactions

• Most of their nonpolar side chains are buried inside

• Nearly all have substantial sections of a-helix and b-sheet

Myoglobin and Hemoglobn

• Myoglobin is a protein (globin) containing a single hemeunit, which stores oxygen in cells (especially muscles)

• Hemoglobin is a multimeric protein with four sub units

• May occur intracellulary or extracellularly

• Extracellular hemoglobin has a very high molecular weight

Both Proteins Contain a Prosthetic Group

• Porphyrins: Metal complexes

derived from porphyrin

• Many respiratory pigments are

designed around the porphyrin

molecule

• After the two H atoms bound

to N are lost, porphyrin is a

tetradentate ligand

• Two important porphyrins are

heme (Fe2+) and chlorophyll

(Mg2+)

Prosthetic Group

• Four N atoms from the porphyrinring are attached to the Fe2+

center

• Fifth coordination site is occupied by a base (Histidine), of the globinprotein

• Sixth coordination site can be occupied by

• O2 (oxyhemoglobin)

• H2O in (deoxyhemoglobin)

• CO in (carboxyhemoglobin)

• Role of the globin

• Prevent oxidation of Iron

• Reduce affinity to CO

Factors that Effect Oxygen Binding

• Binding of oxygen to Hb displays co-operativity

• Number of factors can influence binding of oxygen

• Organic Phosphates (DPG/BPG)

• Temperature

• pH• CO2

Genetic Basis of Hemoglobin

Composition of human hemoglobin chains at different life stages:

Embryo : z2e2, a2e2

Fetus : a2g2

Adult : a2b2 (97%), a2d2 (2-3%), a2g2 (1%)

• Encoded by a multi-gene family a-globin family: a, z

b-globin family: b, d, e, g

• Mutations of the Hb genes can result in diseases such as• Sickle cell anaemia• Thalassemia

Sickle-Cell Anemia•Results from a single mutation in the beta chain

Glu Val

• (-) charge is changed to a nonpolar (hydrophobic) group

• This site of mutation is at the surface of the protein in the deoxy form of hemoglobin.

• This results in the beta chains ‘sticking’ together in the deoxy form

Immunoglobulins

• The antibody molecule comprise of the immunoglobulin

domain

• Immunoglobulin domain comprise of a 100 –110 aa held

together by intra-chain disulfide bonds that forms a

compact loop within the chain (globular domain)

• 2 Heavy chains

• 2 Light chains

• The four chains are held together by disulphide linkages

• The quaternary structure is Y shaped with three arms

Functions of Antibodies

Enzymes: Biological Catalysts• Almost all reactions in cells are catalyzed by

enzymes

• Generally most enzymes are proteins

• However RNA can also catalyse reactions (Ribozymes)

• Enzymes accelerate reactions by lowering the free energy of activation

• Enzymes do this by binding the transition state of the reaction better than the substrate

• Transition state is halfway between substrate structure and product structure

Lect. 11-

62

Enzymes lower ∆G‡ (Activation energy) but do not affect ∆G (standard state free energy) for a reaction

How Enzymes Aid in the Catalytic Process

• Bind substrates

• Lower the energy of the transition state

• Directly promote the catalytic event

• Either through acidic or basic side chains that promote addition or removal of protons

• Or through holding ions in correct position to participate in the catalysis

• Release the products

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Cofactors

• In addition to the protein part, many enzymes also have a nonprotein part called a prosthetic group or a cofactor

• The protein part in such an enzyme is called an apoenzyme, and the combination of apoenzyme plus cofactor is called a holoenzyme.

• Only holoenzymes have biological activity; neither cofactor nor apoenzyme can catalyze reactions by themselves

• Cofactors form an intricate part of the active site and play a direct chemical role in the chemistry of the reaction

Cofactors

• A cofactor can be either an inorganic ion or an organic molecule, called a coenzyme

• Many coenzymes are derived from vitamins, organic molecules that are dietary requirements for metabolism and/or growth

• Nicotinamide adenine dinucleotide (NADH)

• Flavin adenine dinucleotide(FADH)

• Heme group

• Metal atoms e.g. Zn++Me

Me

N

OH

OH

N

OH

N

O

O

N H

O

P

O

OHO

P

O

O

HO

O

OH

N

HO

N

N

NH 2

N

SS

R

R

S

R

R

FADH

HO

N O

OH

N

N

O

P

NH 2

N

O

O OH

P

O

O

O

O

HO

N

OH

NH 2

O-

+

R

R

S

R

R

S

R

R

NADH

66

Classification of Enzymes

Class Reactions catalyzed

Oxidoreductoases oxidation-reduction Transferases transfer group of atoms Hydrolases hydrolysis

Lyases add/remove atoms to /from a double bond

Isomerases rearrange atoms Ligases combine molecules using ATP

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Enzyme Action: Lock and Key Model

• An enzyme binds a substrate in a region called the active site

• Only certain substrates can fit the active site

• Amino acid R groups in the active site help substrate bind

• Enzyme-substrate complex forms

• Substrate reacts to form product

• Product is released

68

Enzyme Action: Induced Fit Model

• Enzyme structure flexible, not rigid

• Enzyme and active site adjust shape to bind substrate

• Substrate molecule induced to take up a configuration approximating the transition state

• Shape changes also improve catalysis during reaction

• Increases range of substrate specificity

69

Lock and Key Model vs. Induced Fit Model

E + S ES complex E + P

70

Factors Affecting Enzyme Action: Temperature

• Little activity at low temperature

• Rate increases with temperature

• Most active at optimum temperatures (usually 37°C in humans)

• Activity lost with denaturation at high temperatures

71

Factors Affecting Enzyme Action

Optimum temperature

Reaction

Rate

Low High

Temperature

72

Factors Affecting Enzyme Action: Substrate Concentration

• Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant)

• Maximum activity reached when all of enzyme combines with substrate

73

Factors Affecting Enzyme Action: pH

• Maximum activity at optimum pH

• R groups of amino acids have proper charge

• Tertiary structure of enzyme is correct

• Narrow range of activity

• Most lose activity in low or high pH

74

Optimum pH

Reaction

Rate

3 5 7 9 11

pH

Factors Affecting Enzyme Action: pH

Gene RNAProteinEnzyme

InactiveEnzyme

Functional Enzyme

ActiveEnzyme

Modification

• This can be achieved through two mechanisms

Regulation of synthesis

Regulation of degradation

• Synthesis can be regulated at two levels

Transcription regulation

Translation regulation

Regulation at Enzyme Function

Allosteric Regulation

• Enzyme is regulated by binding of specific stimulatory or inhibitory protein

• Eg. Calcium-calmodulin

• Regulatory subunit of cAMP dependent protein kinase

Target Ca++/CAM dependent

protein KinaseCa++ / Calmodulin

Activated Ca++/CAM dependent

protein Kinase

Stimulation & Inhibition by Control Proteins

• Some enzymes are produced as inactive Zymogens or proenzymes

• The active site of these enzymes are masked by a part of the molecule

• Cleavage of the masking portion by spontaneous degradation or other proteolytic enzymes leads to exposure of the active site and therefore activation

Digestive enzymes: Procarboxypeptidase, Pepsinogen, Trypsinogen,

Proteolytic Cleavage

This result in conformational changes of the enzyme

• Covalent modification is targeted at a R group of one of the amino acids moieties of the protein

• Phosphorylation at serine, threonine or tyrosine residues eg. Glycogen phosphorylase

• Adenylylation at tyrosine residues eg Glutamine synthase

• Carboxymethylation at aspartic or glutamic acid residues

Reversible Covalent Modification

• Some enzymes and enzyme complexes have fixed

locations within the cells or body

• Nucleus: DNA replication, synthesis of tRNA

and mRNA and some nuclear proteins

• Ribosomes: Protein synthesis

• Chloroplast: Photosynthesis

• Liver: Fatty acid metabolism, Gluconeogenesis,

Glucose metabolism, Glycogen synthesis

• Adipose tissue: Fat metabolism

Compartmentalization within Organelles or Organs

Enzyme Kinetics

• For a given amount of enzyme the relationship between reaction velocity and substrate concentration

E + S ES Pk1

k-1

k2

Vmax [S]Vinit =

KM + [S]

Michaelis – Menton rate equation

Enzyme Kinetics

• Lineweaver-Burk equation and plot allows us to determine Vmax and Km

83

Enzyme Inhibition

• Cause a loss of catalytic activity

• There are FOUR types of enzyme inhibition:

1. Irreversible

2. Competitive

3. Non-Competitive

4. Uncompetitive

Irreversible Inhibition

• A compound interferes with the active site so as to disable it

• Commonly it is done by forming a stable covalent adduct with the enzyme

• May also block substrate access to site

• Almost all are toxic substances

Diisopropyl

Fluorophosphate

(DIFP)

Competitive Inhibition

When an unreactive molecule bind to an enzyme’s active site and compete with the substrate to bind enzyme

Vmax remain unchanged

Km appear to increase

* Increased substrate can overcome inhibition

Non-Competitive Inhibition

An inhibitor that binds to the enzyme, but not at the active site. In this case the inhibitor is not competing for the active site

- Binding distorts the enzyme and reduces its activity e.g. allosteric regulation of the enzyme

This form of inhibition causes:

Vmax to drop

Km remains unchanged

* increased substrate cannot overcome a non-competitive inhibitor

Uncompetitive inhibition

• Substrate binding to enzyme is not inhibited

• Inhibitor binds to the ES complex occurs

• ESI complex is stabilized relative to ES complex so Km is reduced

• ESI complex is non-productive so Vmax is lowered

How enzymes aid in the catalytic process

• Bind substrates

• Lower the energy of the transition state

• Directly promote the catalytic eventEither through acidic or basic side chains

that promote addition or removal of protonsOr through holding ions in correct position to

participate in the catalysisOr by inducing stress that makes bonds labile

• Release the products

biochemistry lecture (proteins)

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