Proteins and enzymes Flashcards
1
Q
Explain the structure of amino acids.
A
- They are biopolymers composed of amino acids
- Amino acids composed of amino group (-NH2), carboxyl group (-COOH), (-H) and a R group
- 20 different amino acids, vary in structure depending on the R group
- Amino acids are known as 2-amino acids, since R group and amino group attached to second carbon
- Amino acids are coded by genetic material, are universal (due to universal genetic code)
- Proteins form by condensation reactions
2
Q
Why are amino acids amphoteric?
A
- All are amphoteric, they can behave as acids and bases in chemical reactions
- The carboxyl group (-COOH) is acidic and can ionise to release H+ ion
- The amino group (-NH2) is basic, nitrogen can donate pair of electrons and accept H+ ion
- R group can provide acid base activity
3
Q
What are zwitterions?
A
- Since amino acids are amphoteric, they can exist as zwitterions, they contain both positive and negative charge (dipolar ion)
- Amino acids exist in zwitterion form in a pH range close to neutral
- Buffer solutions maintain a pH range close to neutral, hence amino acids are usually in zwitterion form
4
Q
How do zwitterions behave in different pH ranges?
A
- In acidic media, they act as bases, COO- group gains H+ to form COOH (acid becomes neutral)
- NH3+ group remains, making it a cation
- In basic media, zwitterion acts as acid, NH3+ releases a H+ to from NH2 and COO- remains, making it an anion (base becomes neutral)
5
Q
What is the isoelectric point?
A
- The pH at which an amino acid either becomes an acid or a base
- Isoelectric point: pI
- Each amino acid has unique pI, depending on properties of R group
6
Q
What pI do the R groups have?
A
- R groups that have no acidic or basic properties and are non-polar have pI of 6
- Polar R groups have lower pI than non-polar R groups
- R groups that have acidic functional groups have an even lower pI
- R groups with basic functional groups and polar have higher pI
7
Q
Explain the melting point properties of amino acids.
A
- All amino acids are crystalline solids, have high melting points
- Because of the ionic charges present on zwitterions
- Presence of opposite charges allow strong electrostatic attractions between amino acids
- Normal amino acids (not in zwitterion form) have a lower attraction and melting points
- R groups can further increase or decrease melting points
- Acidic or basic groups can be ionised, further ionic charges, increases melting point
- R groups with strong intermolecular forces have high melting point
8
Q
Explain the solubility properties of amino acids?
A
- Charges in zwitterions allow high solubility in aqueous solution in biological environments
- Charged groups allow strong ion-diple attractions in polar solvents, but weak attractions in non-polar solvents
- If weak attraction in non-polar solvent, amino acids form stronger attractions with each other, low solubility
- Highly polar R group show higher solubility
- R groups that are non-polar show lower solubility
9
Q
How do polypeptide chains form?
A
- Amino acids joined together by a condensation reaction
- NH2 loses a H+ ion and COOH from another amino acid loses -OH-
- Forms amide linkage, peptide bond and H2O
- No. of water molecules removed always one less than number of amino acids joined
- Only a polypeptide chain with three-dimensional shape can be called a protein
- The end of the polypeptide chain with amino group is the N terminal, the end with carboxyl group is C terminal
10
Q
How is a polypeptide chain broken down?
A
- Hydrolysis reaction, water added with help of enzymes to catalyse the reaction
11
Q
Explain the primary structure of a protein.
A
- Sequence of amino acids residues bonded together in the polypeptide chain by covalent bonds
- Sequence coded by genetic material
- If mistake in genetic code, result in wrong amino acid sequence, cannot perform its function
12
Q
What are amino acid residue?
A
- Remains of amino acids after polymerisation reaction
- Since condensation results in loss of a hydrogen and hydroxide, the molecule is no longer complete
13
Q
Explain the secondary structure of a protein.
A
- Includes coiling or folding of polypeptide chain
- Can take up alpha helix (coiled) or beta pleated sheet (folding)
- These structures held together by hydrogen bonds from distant amino acid residues
14
Q
What is an alpha helix?
A
- Coiling of the backbone of polypeptide chain, R group extend outward from the spiral coil
- Typical helix contains 10 amino acid residues
- Held together by strong hydrogen bonds between carboxyl and amino groups
15
Q
What is a beta sheet?
A
- Alternating linear folds of an extended backbone
- Gives pleated formation (paper fan)
- This allows beta sheets from different parts of the polypeptide chain to align together
- Held by hydrogen bonds
- Opposite orientation, maximises interaction of hydrogen bonding
- Usually 2 to 10 amino acids in length
16
Q
Explain the tertiary structure of a protein.
A
- Includes the three-dimensional globular structure of the protein
- Type of interactions: disulfide bridges, alpha helices, beta sheets
- Polypeptide folds to allow better solubility in aqueous solution: polar R groups exterior, non-polar R groups interior
- Intermolecular forces of attraction between two polar R groups and between two non-polar R groups
- Acidic and basic R groups that undergo ionisation, can have electrostatic forces of attraction, much stronger than those of uncharged R groups
17
Q
What are disulfide bridges?
A
- Present in tertiary level between cysteine amino acid residues
- When two amino acids containing thiol group (-SH) are joined, sulfur atoms can form covalent bond through oxidation reaction, releases 2 H+ and 2 e-
- Amino acids can only form disulfide bridge when thiol group present
18
Q
Explain the quaternary structure of a protein.
A
- Combination of more than one polypeptide chain
- Can contain one or multiple chains and other species such as transition metals
- Intermolecular forces hold polypeptide chains together, not chemical bonds
19
Q
What is the role of a protein?
A
- When a polypeptide chain folds into a three-dimensional shape, it can now play a role in metabolic processes
- Some act as structural components of cells or act as enzymes, chemical messengers to transport molecules or biological catalysts
- Act as energy source of consumers
20
Q
What are the characteristics of globular proteins?
A
- Spherical shape, determined by their tertiary structure and intermolecular forces between R groups
- Soluble in aqueous solutions due to presence of polar R groups in the exterior of protein and non-polar R groups in interior
- Act as chemical messengers (hormones), catalysts (enzymes) and transport molecules
- Sensitive to high temperatures, may lead to denaturing (unfolding) of protein, weaken forces
21
Q
What are the characteristics of fibrous proteins?
A
- A.k.a scleroproteins, consist of long linear bundles of polypeptide chains, covalently bonded or held together by disulfide bridges or hydrogen bonds
- Insoluble in aqueous solution due to presence of exposed non-polar R groups
- Form the basis of structural elements in an organism, due to their strength or storage proteins
- Less sensitive to high temperatures, compared to globular proteins, fibres held together by covalent bonds and not intermolecular forces
22
Q
Give two examples of globular proteins and their function and two fibrous proteins.
A
- Globular: insulin, hormone, responsible for regulating level of glucose in the blood
-Globular: pepsin, enzyme, digests proteins in the stomach, makes amino acids available to make new proteins - Fibrous: collagen, structural, soft and flexible proteins form hair, fingernails and blood vessels
- Fibrous: casein, storage, stores amino acids, carbohydrates, calcium , phosphorous in milk from mammals
23
Q
What do enzymes do?
A
- Globular proteins can act as enzymes, they increase rate of reaction by providing an alternative pathway with lower activation energy
- Controls timing or frequency of the reaction
24
Q
Explain the role of an enzyme in a reaction.
A
- If they act as homogeneous catalysts, same phase as other species in reaction
- Provide surface for the reaction to take place
- Shape of enzymes allows interaction with other specific compounds (substrates)
- Substrate is complementary to the active site of the enzyme, forms temporary complex for the chemical reaction to take place
25
What are the two enzyme-substrate theories?
- 'Lock and key', substrate is complementary to shape of active site of enzyme, R groups interact
- Substrate broken apart, two products leave active site, without changing the enzymes molecule
- 'Induced fit', some enzymes function by changing their shape once the substrate binds to the active site, globular proteins are folded by intermolecular forces, exhibit some flexibility
26
How do you measure the enzymatic activity?
- Concentration of enzyme or substrate, temperature, pH
- Changing any of these can result in increase or decrease of enzyme activity
27
What effect does concentration have on enzymatic activity?
- Compare initial rate of product formation over time
- Concentration of enzyme directly affect rate of chemical reaction, more enzymes present
- Lower substrate concentration, increases rate of reaction, since it matches enzyme concentration
- When substrate concentration is higher than enzyme, not enough enzyme to catalyse, rate of reaction approaches maximum
27
What effect does concentration have on enzymatic activity?
- Compare initial rate of product formation over time
- Concentration of enzyme directly affect rate of chemical reaction, more enzymes present
- Lower substrate concentration, increases rate of reaction, since it matches enzyme concentration
- When substrate concentration is higher than enzyme, not enough enzyme to catalyse, rate of reaction approaches maximum
28
What effect does temperature have on enzymatic activity?
- Increase temperature, increase rate
- Since enzymes are globular proteins, heat easily disrupts intermolecular forces, causes enzyme to denature
- Denaturation makes enzyme lose its active site, can no longer perform, hence have optimal temperature
29
What effect does pH have on enzymatic activity?
- Changes in PH can also denature enzymes
- For many in aqueous solutions, pH close to neutral is optimal
- Significant changes in PH can cause amino acid residues with acid-base activity to gain or lose protons, change shape and interactions with other amino acid residues
- pH can be maintained by buffer system
30
What effect do heavy metal ions have on enzymatic activity?
- Certain heavy metal ions have the ability to bind to an enzyme, like a non-competitive inhibitor, decrease in enzyme activity
- Metals: lead (II) and mercury (II), cause immediate and long-term health effect
- Silver, lead, mercury and copper ions can bind to thiol group on cysteine amino acid residues
- Bond formed is covalent, binding or metal is permanent and strong
31
What is chromatography?
- Method use to analyse polypeptides to determine the composition and sequence of amino acid residue
- It separates a mixture to determine number of analytes, relative quantity of analytes, identify the type of analyte
- Separation could be based on polarity of R groups or by size of amino acid
32
How do you perform paper chromatography?
- Mixture is applied as a small spot at bottom of paper
- Bottom of paper suspended in a solvent which travels up by capillary action
- If analyte of mixture attracted to solvent, they will travel up the paper of solvent
- Analytes can be identified if paper chromatography has already been performed
- Distance traveled by analyte, relative to distance traveled by solvent, used to determine Rf value
- Rf: Distance traveled by solute/Distance traveled by solvent
33
What is two-dimensional paper chromatography?
- If the R groups are similar, this method is used to ensure complete separation of all amino acids
- A sequence of two separations with two different solvents (one polar, one non-polar)
- Same method as regular, yet the analytes separate in single column based on degree of interaction with solvent
- Paper turned 90°, making row or spots new origin, place in new solvent
- To see where the amino acids have traveled, locating agent used, reveals the location e.g. ninhydrin (reacts with nitrogen of amino acid)
34
What is gel electrophoresis?
- Method used to separate a mixture of proteins e.g. blood or urine or separating multiple polypeptide chains from a quaternary structure
- Applies samples of protein mixture to cavities cut into a semi-firm gel and applying an electric current
- Polypeptide chains with negative ionic charges, causes them to travel at rate dependent of size, shape or amount of charge
- Separates polypeptides based on size, shape and charge
35
What are the two different methods of gel electrophoresis?
- Native: protein molecule retains its folded structure, separation based on size and shape
- Smaller travel faster
- Denaturing: proteins first denatured with heat or chemical detergent, lose shape and return to linear polymer
- Travel through gel based on only size, not both, better separation
- Use samples of known polypeptides, provide reference
- Looks life a ladder
