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
Q
What are the two enzyme-substrate theories?
A
- ‘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
Q
How do you measure the enzymatic activity?
A
- Concentration of enzyme or substrate, temperature, pH
- Changing any of these can result in increase or decrease of enzyme activity
27
Q
What effect does concentration have on enzymatic activity?
A
- 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
Q
What effect does concentration have on enzymatic activity?
A
- 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
Q
What effect does temperature have on enzymatic activity?
A
- 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
Q
What effect does pH have on enzymatic activity?
A
- 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
Q
What effect do heavy metal ions have on enzymatic activity?
A
- 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
Q
What is chromatography?
A
- 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
Q
How do you perform paper chromatography?
A
- 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
Q
What is two-dimensional paper chromatography?
A
- 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
Q
What is gel electrophoresis?
A
- 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
Q
What are the two different methods of gel electrophoresis?
A
- 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
