Factors affecting enzyme reactions

Substrate concentration

• At low substrate concentration, increasing the substrate concentration will result in a proportional increase in the rate of reaction.
• As there are sufficient active sites, the increase in substrate concentration will increase the frequency of effective collisions between the enzyme and substrate molecules, increasing the rate of formation of enzyme-substrate complex.
• However, at high substrate concentration, increasing the substrate concentration will no longer speed up the reaction.
• All the active sites of the enzyme molecules will be occupied at any given moment.
• Any added substrates have to ‘wait’ until existing enzyme-substrate complexes are dissociated and the active sites become available for binding.
• The rate of reaction can only be increased with the addition of enzymes.

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Enzyme concentration

• At low enzyme concentration, increasing the enzyme concentration will result in a proportional and linear increase in the rate of reaction.
• The increase in enzyme concentration provides more active sites which the substrate molecules can bind to, increasing the rate of effective collision and the formation of enzyme substrate complex.
• However, at high enzyme concentration, increasing the enzyme concentration will no longer have effect on the reaction as all the substrate molecules would already have been converted into their products.
• The rate of reaction can only be increased with the addition of substrates.

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Temperature

• Enzymes work best at their optimum temperature, which is usually 40°C.
• Below the optimum temperature, the rate of reaction increases linearly with the increase in temperature.
• As the temperature increases, kinetic energy of the molecules increase. This increases the rate of effective collisions between enzyme and substrate molecules and the formation of enzyme-substrate complex.
• Beyond the optimum temperature, the rate of reaction decreases even though the frequency of collisions increases.
• Thermal agitation of the enzymes break the hydrogen bonds, ionic bonds and hydrophobic interactions that stabilize the specific 3D conformation of the enzyme.
• The enzyme becomes denatured as the shape of the enzyme's active site is altered and is no longer complementary to that of the substrate.

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pH

• Enzymes work best at their optimum pH level.
• A change in pH level will result in the alteration of the ionic charge of the R groups of the amino acid residues.
• This breaks the ionic bonds and hydrogen bonds that are responsible for maintaining the specific 3D conformation of the enzyme.
• The enzyme becomes denatured as the shape of the enzyme's active site is altered and is no longer complementary to that of the substrate.

Image taken from: http://alevelnotes.com/content_images/i72_enzyme_ph_graph.gif

Enzymes

What are enzymes?

• large biological molecules which catalyse chemical reactions in living organisms
• responsible for the metabolic processes which sustain life
• enzymes can be proteins or ribonucleic acid (RNA)

Properties of enzymes:

• lower the minimum amount of energy needed to start a reaction (activation energy)
• has active site of specific shape, which is complementary to its substrate 
• does not alter properties of end products of reaction
• highly efficient in small amounts
• denatured easily due to high heat

How do enzymes work?

1. After an effective collision between a substrate and an enzyme (where the substrate binds to the active site of the enzyme), an enzyme-substrate complex is formed. 
2. The substrate molecule is held in the active site by interactions such as hydrogen and ionic bonds between the R groups of the amino acids and the substrate molecule. 
3. Enzyme catalyses conversion of substrate to the end product. 
4. The alteration of chemical conformation results in the product being released from the active site since it is no longer complementary to the structure of the active site. 
5. The active site is then available for other substrates to bind to it.

"Lock and Key" Hypothesis

• there is an exact fit between the substrate and the active site of the enzyme
• the enzyme (lock) has a unique shape complementary to the substrate (key)
• enzymes are very specific; only substrates that are exactly complementary to its active site are able to bind with it

image taken from: http://katysstudynotes.files.wordpress.com/2010/11/enzymes.png

Induced Fit model

• active site of enzyme is flexible 
• active site is able to mould itself around the substrate to make the fit better
• active site returns to original shape after the products are released 

Illustration of the induced fit model

image taken from: http://zebrafish.umdnj.edu/Pre-Enrollment/Resources/Biochemistry/Enzymes%20and%20Metabolism/Images/enzyme_fit_diagram.png

Food Tests

Benedict's Test for Reducing Sugars

• Pour 2cm³ of solution sample solution into a test tube and add an equal volume of Benedict's solution. 
• Shake the mixture.
• Heat test tube in a boiling water bath for 5 minutes.
• Record observation after 5 minutes.

Colour of precipitate and the corresponding amount of reducing sugar present

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Biuret's Test for Proteins

• Add 2cm³ of sample solution to a clean test tube and add 2cm³ of dilute sodium hydroxide solution.
• Shake the mixture.
• Add 1% copper (II) sulphate solution, drop by drop. Shake mixture after every drop and observe the colour change. 
• If mixture remains blue, no protein is present.
• If mixture turns violet/purple, protein is present. 

Iodine Test for Starch

• Add 2cm³ of sample solution to a clean test-tube, followed by a few drops of iodine solution.
• Observe the colour change.
• If mixture remains brown, no starch is present.
• If mixture turns blue black, starch is present.

Ethanol Emulsion Test for Fats

• Add 2cm³ of sample solution to a clean test tube and add 2cm³ of ethanol.
• Shake mixture thoroughly.
• Add 2cm³ of water into the test tube and shake. 
• Record the observation.
• If mixture remains clear, no lipid is present.
• If a white emulsion was obtained, lipid is present. 

Condensation and Hydrolysis

Condensation and hydrolysis are reverse processes of each other.

Condensation (or dehydration synthesis) is a chemical process which by 2 molecules are combined to make a larger molecule through the loss of water. It is the process which by biomolecules are formed.

Hydrolysis is a chemical process which by a macromolecule is broken down into smaller molecules by the addition of water. Often, enzymes are needed to carry out hydrolysis, as water alone is insufficient.

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Biomolecules — Lipids

What are lipids?

• lipids are insoluble in water
• contain C, H and O
• contain less oxygen than carbohydrates
• made up of fatty acids and glycerol

What are fatty acids?

• contain hydrophobic "tail" of a long hydrocarbon chain, and hydrophilic "head" which is a carboxyl group
• can be saturated (no double bonds, no kinks) or unsaturated (double bonds and kinks in fatty acid tail)

Classification of lipids

• simple lipids
• compound lipids
• steroids and sterols

Simple Lipids

• formed by joining fatty acid with an alcohol (eg. glycerol)
• fats (eg. triglyceride) are simple lipids
• triglyceride structure: glycerol + 3 fatty acid tails

Compound Lipids (eg. phospholipids)

• found in phospholipid bilayer of cell membrane
• phospholipid structure: phosphate head + 2 fatty acid tails

Biomolecules — Carbohydrates

Carbohydrates are the most abundant biomolecule in nature. 

Function of carbohydrates

• building blocks for larger molecules 
• energy storage — starch (plant) and glycogen (animal)
• structural — cellulose cell wall (plant) and chitin (insects, crabs, shrimps)

The bond between monomers of carbohydrates is known as glycosidic bond.

Monosaccharides

• monomers of carbohydrates
• eg. glucose, fructose, galactose
• all monosaccharides are reducing sugars

Disaccharides

• made up of two monosaccharides

http://i.imgur.com/2Kx8x.jpg

Polysaccharides

• made up of many monosaccharides

What is starch? 

• polymer of glucose molecules
• has two components; amylose and amylopectin
• both fit together to form a complex 3-dimensional structure which is insoluble in water
• amylose helices are entangled in the branches of amylopectin molecules
• each amylose chain is coiled into a helix, with six glucose residues for every complete turn of the helix — compact shape making it a complex structure for storage
• amylopectin have many branches

What is glycogen?

• animal equivalent of starch
• found in liver and skeletal muscles of vertebrate animals 

Starch and glycogen (energy stores)

• their molecules have many side branches where glucose molecules can be removed from their tips (by enzymes)
• their insolubility stops them interfering with osmosis
• their compactness provides an efficient way to store lots of glucose for future cellular respiration

Cellulose (long, unbranched chain)

• most abundant organic molecule on Earth
• major component of cell wall in plants
• made from long, straight unbranched chains of glucose
• chains cross-linked by H-bonds which holds them tightly together (excludes water)
• chemically very inert and insoluble 
• many molecules form strong fibrils
• only some bacteria, fungi and a very small number of animals can secrete cellulase enzymes

Biomolecules — Proteins

Amino Acids:

The basic building blocks of proteins are amino acids. Amino acids are the monomers which form polypeptide and proteins.

Properties of Proteins:

• sensitive to pH and heat
• shape determines function
• enzymes are special group of proteins
• contain H, N, C and S
• made up of amino acids

There are about 20 essential amino acids found in proteins, and they differ in their R group. 

Amino acids join together to form a polypeptide through the process called condensation.

Two amino acids are joined by a peptide bond to form a dipeptide via condensation. Water is formed as a by product. 

The reaction is between an amino group and a carboxyl group. 

Continued condensation leads to the addition of more amino acids and thus resulting in a polypeptide chain.

 

Structure of proteins:

1. Primary structure --> specific linear order which amino acids join to form a polypeptide chain
2. Secondary structure --> regular folding of segments by hydrogen bonds (α-helix and β-pleated sheet)
3. Tertiary structure --> (only for globular proteins) overall shape of polypeptide resulting from interactions* between the R group 
4. Quaternary structure --> association of 2 or more polypeptide chains 

*Bonds that form tertiary structures:

• hydrogen bonds
• ionic bonds
• disulfide bonds
• hydrophobic interaction

Classification of proteins (based on structure):

• Globular --> compact and spherical structure which is soluble
• Fibrous --> long polypeptide chains twisted around each other, forming long fibres that provide tensile strength, insoluble