HMM1414 Chapter 2

8/9/2019 HMM1414 Chapter 2

1/58

HMM1414/Chapter 2

CHAPTER 2

CHEMICAL FOUNDATION OF LIFE2.1 Properties of Water

(Refer Campbell page 47 53)

Origin of life - water.

Terrestrial organisms also tied to water:

o Cells surrounded by water.o Cell content: 70 95% water.

o Reactants.

2.1.1 Polarity of Water & HydrogenBonding

Water- po la r mo lecu le Two hydrogen atoms form po la r cova len tb o n d with an oxygen atom. Oxygen more electronegative than hydrogen.

Oxygen 2 partial negative charge ( -). Hydrogen - partial positive charge ( +). Attraction between water molecules:

Negative regions attracted to positive regions,

forming h y d r o g e n b o n d s . Maximum offour bonds per molecule.

(Refer Figure 3.2, Campbell page 47)

1

8/9/2019 HMM1414 Chapter 2

2/58

HMM1414/Chapter 2

2.1.2 Heat Capacity Spec i f ic hea t - amount of heat that must beabsorbed or lost for 1 g of a substance to change

its temperature by 1C.

Specific heat of water = 1 cal/g/C.

Water resists/minimizes temperature

fluctuations because of its high specific heat.

Absorbs or releases relatively large quantity of

heat for each degree of temperature change.

Importance of high specific heat:

Water heats up more slowly & holds its

temperature longer.

Major component of living things.

Medium for biochemical reactions.

Aquatic environments have relatively stable

temperatures.

2

8/9/2019 HMM1414 Chapter 2

3/58

HMM1414/Chapter 2

2.1.3 Heat of Vaporization Heat of vaporization quantity of heat aliquid must absorb for 1 g of it to be converted

from liquid to gas.

Water - high heat of vaporization

As a liquid evaporates, the surface of the liquid

that remains behind cools - evaporativecooling.

Importance:

Cooling mechanism during sweating &

transpiration.

Large amount of heat is lost during evaporation

with only minimal loss of water from body.

Evaporative cooling moderates temperature in

lakes and ponds.

3

8/9/2019 HMM1414 Chapter 2

4/58

HMM1414/Chapter 2

2.1.4 Density Water less dense as solid than as liquid.

Water expands as it solidifies. In liquid form, not all molecules are H-bonded

to 4 other molecules.

H-bond constantly break & reform - allowsmolecules some freedom of movement.

When temperature falls below 0C, hydrogenbonds are locked into a crystalline lattice.

o Each molecule is hydrogen-bonded to 4other molecules.

o Molecules are further apart from eachother.

o Fewer molecules than an equal volume ofliquid water, making ice less dense.

(Refer Figure 3.5, Campbell, page 51)

As ice melts, some H bonds break, & watermolecules move closer to one another densityincreases.

Maximum density at 4C.

Above 4C, density decreases as waterexpands & the molecules move faster.

Ice is 10% less dense than water at 4C. Thus, ice floats on the cool water below. Importance:

If ice sank, all ponds, lakes, and ocean wouldfreeze solid.

Insulation

4

8/9/2019 HMM1414 Chapter 2

5/58

HMM1414/Chapter 2

2.1.5 Solvent Properties Water - versatile solvent because of its

polarity.

Readily forms H bonds with charged and polar

covalent molecules.

E.g., when a crystal of salt (NaCl) is placed in

water(Refer Figure 3.6, Campbell, page 51)

Na+ oxygen. Cl hydrogen.

Ion surrounded by a hydration shell.

Polar molecules also soluble in water because

they form H bonds with water.

Large molecules can dissolve in water if theyhave ionic and polar regions.

Any substance that has an affinity for water is

hydrophilic(water-loving). These substances are dominated by ionic or

polar bonds. Substances that have no affinity for water are

hydrophobic(water-fearing). These substances are nonionic and have non

polar covalent bonds.

5

8/9/2019 HMM1414 Chapter 2

6/58

HMM1414/Chapter 2

2.1.6 Cohesion and Adhesion Cohesion the binding together of likemolecules, often by hydrogen bonds.

Cohesion among water molecules enables

transport of water against gravity in plants.

Adhesion,attraction between different kindsof molecules, also plays a role - enables water to

adhere to the wall of the vessels.

Surface tension, a measure of the forcenecessary to stretch or break the surface of a

liquid, is related to cohesion.

Water has a greater surface tension because

hydrogen bonds among surface water

molecules resist stretching or breaking the

surface. Importance:

Some animals can stand, walk, or

run on water.

Capillary action

6

8/9/2019 HMM1414 Chapter 2

7/58

HMM1414/Chapter 2

2.1.7 Viscosity

Viscosity a measure of a fluids resistance to

flow.

Water low viscosity.

Importance:

Transport system of living organisms.

Example: blood is mostly water can flow

easily through vessels

Plants depend on flow of water in xylem

& phloem to transport substances.

Less energy used by aquatic organisms

when swimming in water.

2.1.8 Other Properties

Colorless & transparent

Transmission of sunlight possible

photosynthesis for aquatic plants.

Difficult to compress

Structural agent hydrostatic

skeleton.

Involved in many chemical reactions

Raw material for photosynthesis

Digestive reactions breaks down

food molecules by hydrolysis

7

8/9/2019 HMM1414 Chapter 2

8/58

HMM1414/Chapter 2

2.2 The Chemistry of Carbon2.2.1 Definition of Organic Compounds

Organic compounds compound containing

carbon. Cells consist mostly of carbon-based

compounds.

Carbon can form large, complex, and diversemolecules.

Accounts for diversity of biological molecules

great diversity of living things. Proteins, DNA, carbohydrates are composed of

carbon atoms bonded to each other, & to atoms

of other elements - hydrogen (H), oxygen (O),nitrogen (N), sulfur (S), and phosphorus (P).

2.2.2 Structure of Carbon andFormation of Covalent Bonds inCarbon

Carbon atom - 2 electrons in 1st electron shell

& 4 in 2nd shell.

To complete its valence shell, it share

electrons with other atoms in four covalent

bonds.

This tetravalence by carbon makes large,complex molecules possible.

8

8/9/2019 HMM1414 Chapter 2

9/58

HMM1414/Chapter 2

Can form covalent bonds with many different

elements.

The valences of carbon and its partners -

building code that governs architecture of

organic molecules.

Carbon chains form skeletons of most organic

molecules.

Skeletons vary in length and shape.(See Figure 4.5, Campbell, page 61)

Skeletons may have double bonds & may have

atoms of other elements bonded to it.

Variationin architecture of organic moleculescan also be seen in isomers.

Distinctive properties of organic molecules

also depend on molecular components attached

to it.

Functional groups - components oforganic molecules commonly involved in

chemical reactions. The number and arrangement of

functional groups help give each molecule its

unique properties.

(See Figure 4.10, Campbell, page 64 65)

9

8/9/2019 HMM1414 Chapter 2

10/58

HMM1414/Chapter 2

2.2.3 Macromolecules Small organic molecules are joined together to

form macromolecules. Consist of thousands of covalently bondedatoms.

Four major classes: carbohydrates, lipids,proteins, and nucleic acids.

Carbohydrates, proteins, and nucleic acids

polymers. Polymer - long molecule consisting of many

similar or identical building blocks(monomers) linked by covalent bonds.

Monomers connected by covalent bonds bycondensation reaction ordehydrationreaction. Polymers are disassembled by hydrolysis.

10

8/9/2019 HMM1414 Chapter 2

11/58

HMM1414/Chapter 2

2.3 Important Organic Compounds2.3.1 Carbohydrates

Sugars and their polymers. Monosaccharides - single/simple sugars.

Disaccharides - double sugars.

Polysaccharides - polymers ofmonosaccharides.

(a) Monosaccharides

Molecular formula - multiple of the unit CH2O.

For example, glucose - C6H12O6.

Have a carbonyl group (>C=O) and multiplehydroxyl groups (OH).

Classification based on:(1) Location of carbonyl group - aldose orketose. Example: Glucose, galactose, riboses

aldose; fructose,ribulose ketose

(2) Number of carbons in carbon skeleton.

Hexoses 6C sugars - glucose. Pentoses 5C sugars ribose.

Trioses 3C sugars - glyceraldehyde.

(See Figure 5.3, Campbell, page 70)

Monosaccharides in aqueous solutions formrings.


11

8/9/2019 HMM1414 Chapter 2

12/58

HMM1414/Chapter 2

Functions:

1.Major fuel for cellular work.

2.Raw material for synthesis of other monomers.

(b) Disaccharides Form from two monosaccharides viadehydration.

Covalent bond - glycosidic linkage. Maltose - glucose + glucose. (See Figure 5.5 (a),

Campbell, page 71)

Sucrose - glucose + fructose. (See Figure 5.5 ba),Campbell, page 71)

Lactose- glucose + galactose.

(c) Polysaccharides Polymers of many monosaccharides joined byglycosidic linkages.

Functions:

1.Storage.

2. Building materials/structural roles.

12

8/9/2019 HMM1414 Chapter 2

13/58

HMM1414/Chapter 2

(i) Storage PolysaccharidesStarch

Storage polysaccharide of plants.

Monomers (glucose) joined by 14 linkages.

Two forms:

Amylose - unbranched and forms a helix. Amylopectin - branched and more complex.

(See Figure 5.6 (a), Campbell, page 72)

Plants store surplus glucose as starchgranules within plastids, including chloroplasts.

Glycogen Storage polysaccharide in

animals.

Highly branched like amylopectin.

(See Figure 5.6 (b), Campbell, page 72) Stored liver and muscles.

(ii) Structural PolysaccharidesCellulose

Major component of plant cell

wall. Polymer of glucose, but with

different glycosidic linkages.

Difference based on two slightly different ringstructures for glucose: - and -glucose.


Starch: -glucose monomers. (See

Figure 5.7 (b), Campbell, page 73)

13

8/9/2019 HMM1414 Chapter 2

14/58

HMM1414/Chapter 2

Cellulose: -glucose monomers.(See Figure 5.7 (b), Campbell, page 73)

Starch ( 1-4 glycosidic linkages) helicalstructure

Cellulose ( 1-4 glycosidic linkages) straightstructures.

H atoms on one strand can form hydrogenbonds with OH groups on other strands.

Plant cell walls - parallel cellulose moleculesheld together into microfibrils - strongbuilding materials for plants. (See Figure 5.8,

Campbell, page 73) Enzymes that hydrolyze starch

cannot hydrolyze the -linkages incellulose.

Cellulose in human food eliminated in feces asinsoluble fiber.

Some microbes use ce l lu lase todigest cellulose.

Many eukaryotic herbivores, havesymbiotic relationships withcellulolytic microbes. (See Figure5.9, Campbell, page 74)

Some fungi can also digestcellulose.

Chitin In exoskeletons of arthropods.

Glucose monomer has nitrogen-containingappendage. (See Figure 5.10 (a), Campbell, page 74)

Pure chitin is leathery but can be hardened bythe addition of calcium carbonate.

Provides structural support for the cell walls of

many fungi.

14

8/9/2019 HMM1414 Chapter 2

15/58

HMM1414/Chapter 2

Also used to make surgical threads.

15

8/9/2019 HMM1414 Chapter 2

16/58

HMM1414/Chapter 2

2.3.2 Lipid Do not form polymers.

Hydrophobic little or no affinity for water

Consists mostly of hydrocarbons form non-polar covalent bonds.

Most biologically important lipids: fats,

phospholipids, and steroids.

Fats Synthesized from glycerol and fatty acids.

Glycerol: 3C alcohol with a hydroxyl groupattached to each carbon.

Fatty acid: consists of a carboxyl groupattached to a long carbon skeleton.

Non-polar CH bonds in hydrocarbon skeleton

make fats hydrophobic.

Fats separate from water because water

molecules hydrogen bond to one another and

exclude the fats.

Three fatty acids joined to glycerol via ester

linkage, forming triacylglycerol, ortriglyceride.


Fatty acids - same or different.

Fatty acids vary in length (number of carbons)

and in the number and locations of double bonds.

16

8/9/2019 HMM1414 Chapter 2

17/58

HMM1414/Chapter 2

Saturated fatty acid: no C-C double bonds;maximum number of H atom.

Unsaturated fatty acid: one or more C-Cdouble bonds.

Saturated fatty acid - straight chain

Unsaturated fatty acid - has a kink wherever

there is a double bond.

(See Figure 5.12, Campbell. Page 75)

Saturated fats - made from saturated fattyacids.

Example, animal fats - butter.

Solid at room temperature.

Unsaturated fats - from unsaturated fattyacids.

Example: plant (olive oil) and fish fats (cod

liver oil)

Liquid at room temperature = oils.

Kinks prevent the molecules from packing

tightly enough to solidify at room temperature.

Hydrogenated vegetable oils - unsaturated

fats synthetically converted to saturated fats

by addition of hydrogen.

Example: Peanut butter/margarine.

Diet rich in saturated fats - plaque deposits atherosclerosis.

17

8/9/2019 HMM1414 Chapter 2

18/58

HMM1414/Chapter 2

Function of fats1)Energy storage.

1 g of fat stores more than twice as muchenergy as 1 gram of a polysaccharide.

38kJ per g.

Fats compact energy storage as compared

starch which is bulky.

Plants store fats (oils) in seeds; human & other

mammals adipose cells.

2) Adipose tissues cushion (shock absorber) vital

organs - kidneys.

3) Fat (subcutaneous) layer functions as

insulation.

4) Buoyancy for aquatic organisms.

Fats relatively low density

18

8/9/2019 HMM1414 Chapter 2

19/58

HMM1414/Chapter 2

Phospholipids Consists of two fatty acids attached to

glycerol and a phosphate group.


Phosphate group - negative charge.

Additional groups may be attached to the

phosphate groups.

Fatty acid hydrophobic tails.

Phosphate group + attachments - hydrophilic

head. In water, they self-assemble into bilayers with

hydrophobic tails pointing toward interior.


This type of structure = micelle. Major component of all cell membranes.

Example:Lecithin

Steroids Consist of carbon skeleton with four fused

rings.


Cholesterol Precursor from which other steroids are

synthesized.

Example hormones, including vertebrate sex

hormones.

High levels in blood - cardiovascular disease.

19

8/9/2019 HMM1414 Chapter 2

20/58

8/9/2019 HMM1414 Chapter 2

21/58

HMM1414/Chapter 2

Both sexes acne, oily hair & skin, cysts,

jaundice (yellowing of skin), swelling of feet &

ankles, aching joints, bad breath, nervousness,

& trembling.

Risk from taking steroids:

1.Possibility of heart attack & stroke.

2.Increase in anger, hostility, and violent

behavior.

3.Increase risk of getting AIDS from sharing

needles with people that use injectable

steroids.

2.3.3 Protein 50% of dry mass of most cells.

Functions:

1) Structural support

2) Storage

3) Transport

4) Cellular signaling5) Movement

6) Defense against foreign substances

7) Enzymes - catalysts.(See Table 5.1, Campbell, page 78)

21

8/9/2019 HMM1414 Chapter 2

22/58

HMM1414/Chapter 2

Structurally complex molecules: 3-D shape or

conformation.

Built using same set of 20 amino acid

monomers.

Consists of one or more polypeptides. Polypeptides polymers of amino acids.

Amino acids Have carboxyl and amino groups. Four components attached to asymmetric -

carbon atom of amino acid:

(4)

R -carbon|

H N C C = O

| | |

H H H

(3) (1) (2)

1.A hydrogen atom

2.A carboxyl group3.An amino group

4.A variable R group (side chain)

Different R groups characterize the 20

different amino acids.

22

8/9/2019 HMM1414 Chapter 2

23/58

HMM1414/Chapter 2

Physical and chemical properties of R group

determine type of amino acid.


Example:

1.Non-polar (hydrophobic) glycine.

2.Polar (hydrophilic) serine.

3.Charged (ionized):

a)Acidic (negative in charge) Aspartic acid.

b)Basic (positive in charge) Lysine.

Properties of amino acid:

1. Amphoteric has both acidic & basicproperties.

2. Form zwitterions in water carries positivecharge (-NH

3

+) and negative charge (-COO-) at

pH 7.4.

Amino & carboxyl groups ionize in

solution:

-COOH -COO- + H+ (donates H+)

-NH2 + H+ -NH3+ (accepts H+)

Amino acids linked together by peptidebonds forming a polypeptide chain. Polypeptides size - a few to many thousand

monomers.

23

8/9/2019 HMM1414 Chapter 2

24/58

HMM1414/Chapter 2

Each polypeptide has a unique linear sequence

of amino acids.

Levels of Protein Structure Functional protein - one or more polypeptides

twisted, folded, & coiled into a unique shape. Order of amino acids determines three-

dimensional conformation of the protein.

Proteins specific conformation determines itsfunction.

Primary Structure(See Figure 5.20, Campbell, page 82)

Refers to a proteins unique sequenceofamino acids. Example: Lysozyme consists of 129 amino

acids.

Precise primary structure determined by

inherited genetic information.

Secondary Structure(See Figure 5.20, Campbell, page 80)

Refers to repetitive coiling or folding ofthe polypeptide backbone due to hydrogenbond formation between peptide linkages. Typical secondary structures:

24

8/9/2019 HMM1414 Chapter 2

25/58

HMM1414/Chapter 2

Coils ( -helix); or Folds ( -pleated sheets).

25

8/9/2019 HMM1414 Chapter 2

26/58

HMM1414/Chapter 2

Tertiary structure(See Figure 5.20, Campbell, page 83)

Refers to overall shape of polypeptide chainresulting from interactions between the sidechains (R) of the various amino acids.Interaction includes:

Weak interactions:1.H bonds between polar and/or charged areas

2. Ionic bonds between charged R groups

3.Hydrophobic & van der Waals interactions

among hydrophobic (non-polar) R groups.

Strong covalent bonds - disulfide bridges.

Quaternary structure(See Figure 5.20, Campbell, page 83)

Refers to the overall protein structureresulting from the aggregation of two ormore polypeptide subunits.

Collagen: fibrous protein of threepolypeptides, super-coiled like a rope.

Hemoglobin: globular protein.

Four polypeptide subunits: two alpha and

two beta chains.

Each subunit has a heme component withan iron atom that binds oxygen.

26

8/9/2019 HMM1414 Chapter 2

27/58

HMM1414/Chapter 2

Fibrous and Globular Proteins Fibrous protein Elongated molecule

Dominant structure - secondary structure ( -helix or -pleated sheets).

Insoluble.

Plays structural or supportive role in body, &

involved in movement.

Often have regular repeating structures.

Example: Structural proteins

i. Keratin helix of two helices (2 pairs of -helices wound around one another)

consisting of 7 repeating amino acids.

ii. Silk -pleated sheets only (glycine-alanine-serine repeats)

Globular protein Compact and spherical proteins.

Many are folded hydrophobic groups on inside

of molecule &hydrophilic groups face outwards

thus, soluble in water.

Examples: Non-structural proteins enzymes,

transport protein, receptor proteins, and

myoglobin.

27

8/9/2019 HMM1414 Chapter 2

28/58

HMM1414/Chapter 2

Factors determining proteinconformation

1) Primary structure (sequence of amino acids)

2) Physical & chemical conditions of proteins

environment.

Alterations in pH, salt concentration,

temperature, or other factors - denatures aprotein.

Denaturation process in which a proteinunravels & loses its native conformation,

thereby becoming biologically inactive.

3) Organic solvent - polypeptide chain refolds,

causing hydrophobic regions to face outward

towards solvent.4) Heat - disrupts the weak interactions that

stabilize conformation.


28

8/9/2019 HMM1414 Chapter 2

29/58

HMM1414/Chapter 2

2.3.4 Nucleic Acid Amino acid sequence of a polypeptide is

programmed by unit of inheritance, the gene. Gene consists of DNA.

Role of Nucleic Acids

Two types:Ribonucleic acid (RNA): and

Deoxyribonucleic acid (DNA).

DNA provides directions for its own replication.

DNA also directs RNA synthesis and, through

RNA, controls protein synthesis.

The flow of genetic information (The Central

Dogma):

DNA RNA protein

Protein synthesis occurs on ribosomes.

29

8/9/2019 HMM1414 Chapter 2

30/58

HMM1414/Chapter 2

Structure of Nucleic Acids(See Figure 5.26, Campbell, page 87)

Nucleic acids are polymers called

polynucleotides. Polynucleotide is made ofnucleotidemonomers.

Each nucleotide consists of a

(i.) Nitrogenous base

(ii.) Pentose sugar

(iii.) Phosphate group

Pentose sugar +nitrogenous base =nucleoside.

Nucleoside + phosphate = nucleotide

(nucleoside monophsphate)

Nitrogen bases - rings of carbon and nitrogen.

Two types:

(i.) Pyrimidines Have single six-membered ring.

Three types: cytosine (C),thymine (T), and uracil (U).(ii.) Purines

Have a six-membered ring joined to a five-

membered ring.

Two types: adenine (A) and guanine (G).(See Figure 5.26, Campbell. Page 87)

30

8/9/2019 HMM1414 Chapter 2

31/58

HMM1414/Chapter 2

In RNA, pentose sugar is ribose; in DNA it isdeoxyribose.

Difference: Deoxyribose

lacks oxygen atom on

carbon 2.

(See Figure 5.26 (c), Campbell, page 87)

Synthesis of polynucleotides Adjacent nucleotides

joined by covalent bonds

called phosphodiesterlinkages.

Formed between OH group

on 3 C of one nucleotide & phosphate on 5 C

of the next.

This forms repeatingbackbone of sugar-

phosphate units, with

appendages consisting of

nitrogenous bases =

polynucleotide chain.

One end has a phosphate

attached to a 5 C = 5 end.

The other end has a

hydroxyl group on a 3 C= 3

end.


Sequence of bases along a DNA or mRNA

polymer is unique for each gene.

31

8/9/2019 HMM1414 Chapter 2

32/58

HMM1414/Chapter 2

Specifies order of amino acids (primary

structure) of a protein, which in turn

determines its 3-D conformation & function.

32

8/9/2019 HMM1414 Chapter 2

33/58

HMM1414/Chapter 2

The DNA Double Helix(See Figure 5.27, Campbell, page 88)

DNA - two polynucleotide strands doublehelix.

Double helix structure first

proposed in 1953 by James

Watson and Francis Crick.

Sugar-phosphate backbones on outside ofhelix.

Backbones run in opposite 5 -> 3

directions from each other =

antiparallel. Pairs of nitrogenous bases, one from each

strand, connect polynucleotide chains withhydrogen bonds.

Chargaffs Rule of Complementary

base pairing:

Adenine (A) with Thymine (T)

Guanine (G) with Cytosine (C).

2 H bonds between A-T; 3 H bondsbetween C-G.

Strands are complementary.

33

8/9/2019 HMM1414 Chapter 2

34/58

HMM1414/Chapter 2

RNA(Structure of mRNA & tRNA, refer Campbell, page

320 -321)

Single polynucleotide chain.

Nitrogenous base: Adenine (A), Urac i l (U),Guanine (G), & Cytosine.

Pentose sugar : ribose

mRNA messenger RNA:

Delivers information encoded in

genes from DNA to ribosomes, where

information is decoded into protein.

tRNA transfer RNA:

Serves as adapter molecule in

protein synthesis.

Translates mRNA codons into

amino acids.

rRNA ribosomal RNA Structural component of ribosomes.

Forms extensive secondary

structures.

Plays active role in recognizing

conserved portions of mRNAs and tRNAs.

Assist in protein synthesis.

34

8/9/2019 HMM1414 Chapter 2

35/58

HMM1414/Chapter 2

2.4 Techniques of Analysis2.4.1 Chromatography

Physical method to separate & to analyze

complex mixture.

Involves a sample (or sample extract) being

dissolved in a mobile phase (which may be a gas,liquid, or a supercritical fluid).

Mobile phase is then forced through an

immobile, immiscible stationary phase.

Phases chosen so that components of the

same sample have differing solubilities in each

phase. Component which is quite soluble in stationary

phase will take longer to travel through it than a

component which is not very soluble in

stationary phase but very soluble in mobile

phase.

Due to differences in mobilities, sample

components will become separated from each

other as they travel through the stationary

phase.

Paper Chromatography (PC) Stationary phase is liquid soaked into a sheet

or strip of paper.

35

8/9/2019 HMM1414 Chapter 2

36/58

HMM1414/Chapter 2

Mobile phase is a liquid solvent.

Some components spend more time in the

stationary phase others.

Components appear as separate spots spread

out on paper after drying or developing.

Retention Factor, Rf Rf - quantitative indication of how far a

particular compound travels in a particularsolvent.

Rf value - a good indicator of whether an

unknown compound and a known compound are

similar, if not identical.

If Rf value for unknown compound is

close/same as a known compound, then both

compounds are most likely similar or identical.

36
http://en.wikipedia.org/wiki/Image:Cromatography_tank.png

8/9/2019 HMM1414 Chapter 2

37/58

HMM1414/Chapter 2

Rf = Distance the solute moves (D1)

Distance traveled by solvent front (D2).

37

8/9/2019 HMM1414 Chapter 2

38/58

HMM1414/Chapter 2

2.4.2 Electrophoresis Movement of an electrically charged

substance under the influence of an electric

field.

Gel electrophoresis separatesmacromolecules (nucleic acids or proteins) on

basis of their rate of movement through a gel in

an electrical field.

Rate of movement depends on size, electricalcharge, and other physical properties of

macromolecules.


38

8/9/2019 HMM1414 Chapter 2

39/58

HMM1414/Chapter 2

2.4.3 X-ray diffraction X-ray diffraction - scattering of X-rays by atoms

of a crystal.

o Diffraction pattern shows structure of the

crystal.

Can determine 3-D structure of a molecule.

o By determining distances between atoms

of molecules arranged in regular, repeating

crystalline structure.

Technique: X-ray crystallographyo An X-ray beam of light is directed at

crystallized protein.

o Atoms of crystal diffract (deflect) X-rays

into an orderly array.

o Diffracted X-ray exposes photographicfilm, producing a pattern of spots, known as an

X-ray diffraction pattern.

Result:o Using data from X-ray diffraction patterns,

such as a detailed mathematical analysis of

measurements of the spots, as well as amino

acid sequence determined by chemical

methods, a 3-D computer model of the protein

is built.


39

8/9/2019 HMM1414 Chapter 2

40/58

HMM1414/Chapter 2

2.4.4 Centrifugation Cell fractionation - to separate majororganelles of cells so their individual functions

can be studied.

Uses an ultracentrifuge - can spin at up to130,000 revolutions per minute and apply

forces of more than 1 million times gravity

(1,000,000 g).

Fractionation begins with homogenization,gently disrupting the cell.

Homogenate is spun in a centrifuge to

separate heavier pieces into the pellet whilelighter particles remain in the supernatant. As process is repeated at higher speeds and

for longer durations, smaller and smallerorganelles can be collected in subsequent

pellets.

Cell fractionation prepares isolates of specific

cell components.

Enables functions of organelles to be

determined, especially by reactions or processescatalyzed by their proteins.


40

8/9/2019 HMM1414 Chapter 2

41/58

HMM1414/Chapter 2

2.4.5 Microscopy(a) Light Microscope (LM)

In LM, visible light passes through specimenand then through glass lenses.

Lenses refract light, &image is magnified into

eye or onto a video screen.

Vary in magnification and resolving power. Minimum resolution of a light microscope 200 nanometers (nm) = small bacterium.

Can magnify effectively to about 1,000 times

the size of actual specimen.

Various techniques have enhanced contrast

and enabled particular cell components to be

stained or labelled.

Can resolve individual cells, but not internal

anatomy, especially organelles.(See Figure 6.3, Campbell, page 96)

(b) Electron Microscope (EM) Can resolve smaller structures.

EM focuses a beam of electrons through thespecimen or onto its surface.

Have finer resolution.

Resolution could reach 0.002 nanometer (nm),

but practical limit is closer to about 2 nm.

41

8/9/2019 HMM1414 Chapter 2

42/58

HMM1414/Chapter 2

Two types:

(i) Transmission electron microscopes(TEMs) To study the

internal ultrastructure of cells. Electron beam is aimed through a thin section

of specimen.

Image is focused and magnified by

electromagnets.

To enhance contrast, the thin sections are

stained with atoms of heavy metals.

(ii) Scanning electron microscopes(SEMs)

To study surface structures.

Sample surface covered with a thin film ofgold.

Beam excites electrons on samples surface.

These secondary electrons are collected and

focused on a screen.

Result: 3-D image of specimen.

EM reveals organelles impossible to resolve

with the LM.

However, EM can only be used on dead cells.

LM do not have as high a resolution, but can be

used to study live cells.


42

8/9/2019 HMM1414 Chapter 2

43/58

HMM1414/Chapter 2

2.5 Enzymes Catalyst - chemical agent that speeds up rate

of a reaction without being consumed by the

reaction.

Enzyme a catalytic protein speeds up rate ofreaction without being consumed by the

reaction.

Endergonic reaction chemical reactionthat consume energy.o Products formed contain more energy than

reactants.

Exergonic reaction chemical reaction thatreleases energy.

o Products formed contain less energy than

reactants.


2.5.1 Properties of Enzymes1.Reaction catalyzed is specific.

Specificity results from its 3-D shape.

2.

Not destroyed by reactions they catalyzed andare reusable

3.Sensitive to high temperature and pH.

4. Catalyze a reaction in either direction

(reversible).

5. Can be inhibited.

43

8/9/2019 HMM1414 Chapter 2

44/58

HMM1414/Chapter 2

2.5.2 Catalysis and Activation Energy Chemical reaction involves bond breaking &

bond forming.

For this to occur, reactant must absorb energy

from their surroundings.

Formation of new bonds in product

accompanied by release of energy as heat.

Products will have stable shapes withlower energy.

The initial energy that reactants must absorb

before a chemical reaction will start = freeenergy of activation oractivation energy(EA). Activation energy necessary to enable

reactants overcome the energy barrier so that

reaction can proceed.


At the summit, molecules are in an unstable

condition, the transition state.

Reactant may absorb activation energy in the

form of heat from surroundings.

When reactant have absorbed enough energy,

their bonds will break and they become

unstable and, thus, more reactive.

44

8/9/2019 HMM1414 Chapter 2

45/58

HMM1414/Chapter 2

As the products are formed with new, stable

bonding, energy is released to surroundings.

For some processes, EA is not high:

Thermal energy from room temperature is

enough for reactants to reach transition state.

In many cases, EA is high:

Transition state is seldom reached and

reaction hardly proceeds at all.

Reaction will only occur if reactants areheated.

Proteins, DNA, and other complex organic

molecules are rich in free energy and canovercome energy barrier.

However, there is not enough energy in a

typical cell. Most organic molecules cannot make use of

this energy to overcome the barrier.

They may absorb heat, but it would denature

proteins and kill cells.

Enzymes speed reactions by lowering EA.

Transition state can then be reached even atmoderate temperatures.

Enzymes do not change the free energy ( G).

They speed up reactions that would occur

eventually.


45

8/9/2019 HMM1414 Chapter 2

46/58

HMM1414/Chapter 2

2.5.3 Mechanism of Action and Kinetics Substrate - reactant that an enzyme acts on. Enzyme binds to substrate or substrates,

forming enzyme-substrate complex. Catalytic action of enzyme converts substrate

to product or products. Only a specific region of enzyme binds to

substrate by weak chemical bonds- the activesite. Active site is usually formed by only a few

amino acids.


As substrate enters active site, interactionsbetween its chemical groups and amino acids of

enzyme cause an induced fit change in shapeof active site of enzyme so that it binds more

closely to substrate.

Substrates are held in active site by weak

interactions - hydrogen & ionic bonds.

R groups of a few amino acids on active site

catalyze conversion of substrate to product.

Product then leaves the active site.


A single enzyme molecule can catalyze

thousands of reactions a second.

46

8/9/2019 HMM1414 Chapter 2

47/58

HMM1414/Chapter 2

2.5.4 Factors Affecting Enzyme Activity

(1) Substrate Concentration Rate of enzyme-catalyzed reaction increases

as substrate concentration increases until

reaction reaches a maximum rate.

At high substrate concentrations, active sites

on all enzymes are filled. Enzyme is saturated.

To increase productivity at this point - add

more enzyme.

47

Rate

ofr

ea

ction

Substrate concentration

High enzyme concentration

Low enzyme concentration

8/9/2019 HMM1414 Chapter 2

48/58

HMM1414/Chapter 2

(2) EnzymeConcentration

Rate of reaction increases as enzyme

concentration increases, provided no other

factors are limiting.

Relationship only holds when pH, pressure, and

temperature are constant, and substrates are

in excess.

Under these conditions, the more active sitesthere are available, the more substrates can

be converted to products.

48

Rateofr

eaction

Enzyme concentration

8/9/2019 HMM1414 Chapter 2

49/58

HMM1414/Chapter 2

(3) Temperature

Rate of reaction increases with temperature up

to a maximum, the optimum temperature. As temperature increases, collisions between

substrates and active sites occur more

frequently as molecules move more rapidly.

As temperature increases further, weak bonds

are disrupted, and enzyme denatures. Each enzyme has an optimal temperature:

Most human enzymes: 35 40C.

Bacteria in hot springs contain enzymes with

optimal temperatures of 70C or above.

(See Figure 8.18 (b), Campbell, page 155)

(4) pH Most enzymes active at narrow pH range:

Optimal pH: 6 - 8 for most

enzymes.

Many enzymes become inactive, and denatured,

if medium is very acidic or very basic. Substrates no longer fit into active

sites.


(5) Cofactors(6) Inhibitors

49

8/9/2019 HMM1414 Chapter 2

50/58

HMM1414/Chapter 2

Michaelis-Menten Constant, KM Constant represents substrate

concentration required to make reaction go at

half its maximum rate.

Value always same for a particular

enzyme, but varies from one enzyme to another.

Measures affinity of enzyme for its

substrate.

Low KM: high affinity between

enzyme & substrate.

High KM: low affinity substrates

react less readily with enzyme.

50

Rate

ofre

action

Substrate concentration

Maximum rate

maximum rate

Michaelis-Menten constant

8/9/2019 HMM1414 Chapter 2

51/58

HMM1414/Chapter 2

2.5.5 Inhibitors Inhibitors substances which interfere,

reduce, or destroy enzyme activity. Binding of inhibitors to enzymes prevents the

enzymes from catalyzing reactions.

Competitive Inhibitors Inhibitors that resemble substrates and

compete for binding to the active sites.

Binding of inhibitor to active site prevents

enzyme from combining with substrate no

products are generated.

Competitive inhibition overcome by increasing

concentration of substrate.

Reversible.


Noncompetitive Inhibitors Inhibitors that retard enzymatic reactions by

binding to another part of the enzyme. Binding causes enzyme to change shape,

rendering active site less effective at

catalyzing reaction.

Irreversible.

Example: Toxins and poisons.

51

8/9/2019 HMM1414 Chapter 2

52/58

HMM1414/Chapter 2

Allosteric Regulation Refers to binding of a regulatory molecule to a

protein (enzyme) at a site (allosteric site)that affects function of protein at a different

site (active site).

Regulatory molecules - inhibitor or activator.

Binding by these molecules can either inhibit

or stimulate (activate) enzyme activity.

The binding of an activatorstabilizes theconformation of the enzyme so that the active

sites can function or function more effectively.

The binding of an inhibitorstabilizes the

conformation of the enzyme so that the active

sites become inactive or have reduced

catalytic activity.


For example, ATP functions as inhibitor to

catabolic enzymes, inhibiting their activity (so

that less ATP are produced).

ADP functions as an activator of the same

enzymes (so that more ATP are produced)

52

8/9/2019 HMM1414 Chapter 2

53/58

HMM1414/Chapter 2

Feedback Inhibition In feedback inhibition, an early step in a

metabolic pathway is switched off by

pathways final product.

Product acts as an inhibitor of enzyme in that

step.

Feedback inhibition prevents a cell from

wasting chemical resources by synthesizing

more product than is needed.(See Figure 8.21, Campbell, page 157)

53

8/9/2019 HMM1414 Chapter 2

54/58

HMM1414/Chapter 2

2.5.6 Cofactors Non-protein enzyme helpers.

Bind permanently or reversibly to the enzyme.

Two types:

(i) Inorganic cofactors (activators): Example: zinc (Zn2+), iron (Fe2+ or Fe3+), and

copper (Cu+ or Cu2+).

Helps bind together enzyme & substrate or

serve as catalytic centre of enzyme.

(ii) Organic cofactors (coenzymes) Example: Biotin, NAD, FAD, ATP.

Transfers chemical group or atom from

active site of one enzyme to active site of

another enzyme.

Sometimes, function of coenzyme carried by

a non-protein group of atoms attached toenzyme, called prosthetic group. Example: Heme, flavin, and retinal

Transfers chemical groups/atoms from

active site of enzyme to other substance.

54

8/9/2019 HMM1414 Chapter 2

55/58

HMM1414/Chapter 2

2.5.7 Nomenclature of Enzymes (IUB) Produced by the Nomenclature Committee of

the International Union of Biochemistry andMolecular Biology (NC-IUBMB) in consultation

with the IUPAC-IUB Joint Commission on

Biochemical Nomenclature (JCBN).

Enzyme nomenclature is a resource providing

general information on enzyme nomenclature.

Six groups of enzyme:

Group ReactionCatalyzed

Examples1.Oxidoreductase

Oxidation-reductionreactions.

Alcoholdehydrogenase &cytochrome oxidase

2. Transferase Transfer of a functionalgroup from a donormolecule to an

acceptor molecule.

Transaminase &phosphorylases

3. Hydrolases Hydrolytic reaction.Addition of water to, orremoval of water fromsubstrates.

Lipase, amylases,peptidase.

4. Isomerases Conversion of amolecule from oneisomeric form toanother transferatoms from one part of

a molecule to another.

Phosphoglucomutaseisomerase.

5. Ligases Reactions in which twomolecules join in aprocess coupled to thehydrolysis of ATP.

Aminoacyl tRNAsynthetases

6. Lyase Reactions in whichdouble bonds form orbreak, by means otherthan hydrolysis.

Pyruvatecarboxylase & RuBPcarboxylase

55

8/9/2019 HMM1414 Chapter 2

56/58

HMM1414/Chapter 2

2.5.8 Enzyme TechnologyImmobilized EnzymeEnzyme that is physically attached to a solid

support over which a substrate is passed and

converted to product.

Advantages:

i. Multiple or repetitive use of a

single batch of enzymes.

ii. Ability to stop reaction rapidly by

removing enzyme from reaction solution (or

vice versa)

iii. Enzymes are usually stabilized by

bounding.

iv. Product is not contaminated with the

enzyme (especially useful in food andpharmaceutical industries)

Application: example, lactose hydrolysis

Purpose of using immobilized enzymes is to

convert lactose via hydrolysis into glucose

and galactose.

Lactose occurs naturally in both human and

cow's milk. Widely used in baking and

commercial infant-milk formulas.

Problem: Many people are lactose intolerant

-their body is incapable of digesting lactose.

So it must be hydrolyzed into its

monosaccharide components, allowing

56

8/9/2019 HMM1414 Chapter 2

57/58

HMM1414/Chapter 2

digestion, which is the purpose of products

today such as LACTAID.

Biosensor Device that uses specific biochemical reactions

mediated by isolated enzymes, immunosystems,

tissues, organelles, or whole cells to detect

chemical compounds, usually by electrical,

thermal or optical signals.

It is an analytical device which converts a

biological response into an electrical signal

The term 'biosensor' is often used to cover

sensor devices used in order to determine the

concentration of substances and other

parameters of biological interest even where

they do not utilise a biological system directly.

A successful biosensor must possess at leastsome of the following beneficial features:

i. The biocatalyst must be highly specific for the

purpose of the analyses, be stable under

normal storage conditions.

ii. Reaction should be as independent of such

physical parameters as stirring, pH and

temperature as is manageable. This would

allow the analysis of samples with minimal

pre-treatment.

iii. The response should be accurate, precise,

reproducible and linear over the useful

analytical range, without dilution or

concentration.

57

8/9/2019 HMM1414 Chapter 2

58/58

HMM1414/Chapter 2

iv. The complete biosensor should be cheap,

small, portable and capable of being used by

semi-skilled operators.

Schematic diagram showing the main components of abiosensor

The biocatalyst (a) converts the substrate to product.

This reaction is determined by the transducer (b)

which converts it to an electrical signal.

The output from the transducer is amplified (c),

processed (d) and displayed (e).

Documents

HMM1414 Chapter 2