principles of specific binding

Question 1

why is specific protein binding important?

Answer 1

• protein (enzyme) - substrate reactions
• protein inhibition
• drug metabolism/action of drugs.

Question 2

what is a binding site?

Answer 2

a binding site is a region of protein, DNA, or RNA, that a ligand forms a temporary, non-covalent chemical bond with.
binding sites are crevices in the 3D arrangement where a substrate/ligand binds and forms temporary non-covalent bonds with the protein.
binding sites exclude water so is surrounded by non-polar (hydrophobic) amino acids.

Question 3

what is a ligand?

Answer 3

a ligand is a molecule which interacts with a protein, by specific binding.
a ligand can be a nucleic acid, polysaccharide, or lipid.

Question 4

what is the binding of ligands to proteins?

Answer 4

during an enzyme-substrate reaction; the enzyme (protein) must bind to the substrate (ligand) before any chemical changes occur.
any binding of a ligand to a protein is also reversible.
the ligand will bind to the protein at a specific site which has the necessary physical characteristics (shape) to make ligand binding favourable.

Question 5

what are the chemical bonds at the binding site?

Answer 5

non-covalent interactions that keep the ligand attached to the protein.
1. hydrogen bonds
2. ionic bonds
3. hydrophobic interaction
4. van der waals

Question 6

what are the ways a substrate can bind to a proteins active site?

Answer 6

• lock and key model
• induced fit model

Question 7

what are the characteristics of the lock and key model?

Answer 7

the substrate binds to the enzymes active site exactly as they are complementary in shape to each other.

Question 8

what are the characteristics of the induced fit model?

Answer 8

binding of a substrate to an enzymes active sire induces a conformational change in the enzymes active site to bind the substrate more tightly.
reactions can only take place after induced fit has occurred.
the bonds in the enzyme-substrate complex are under stress as the enzyme is not in its natural conformation, this lowers the activation energy required for the reaction to proceed.
induced fit allows the possibility that more than one specific substrate can bind to one enzyme.

Question 9

give an example of the induced fit mechanism.

Answer 9

• hexokinase
• citrate synthase

Question 10

what is a conserved region?

Answer 10

a conserved region/sequence refer to identical or similar sequences of DNA/RNA or amino acids that occur in different (or the same) species over generations.
a conserved sequence demonstrates either no changes, or trace changes in their composition over generations.

Question 11

what does a significant sequence similarity between two molecules suggest?

Answer 11

a significant sequence similarity between two molecules suggests links between common ancestors and therefore likely the same evolutionary origin, with the protein having the same 3D structure, function and mechanism.

Question 12

how are amino acid residues located in binding sites?

Answer 12

amino acid residues in binding sites can be located by a variety of techniques, that are either structural or chemical.
* structural > X-ray crystallography, and the use of structural analogues.
* chemical > chemical modification of amino acid residues, and the varying effect of pH on enzyme activity.

Question 13

what is required for X-ray crystallography?

Answer 13

• a source of X-rays
• protein in a crystal form
• the proteins primary structure.

Question 14

what is the process of X-ray crystallography?

Answer 14

X-ray crystallography involves bombarding a crystal of the protein with X-rays, then analysing the resultant diffraction pattern caused by atoms in the protein scattering the X-rays.

Question 15

what are structural analogues used for?

Answer 15

the use of structural analogues have played a crucial role in our understanding of the interactions within the binding sites.

Question 16

what are structural analogues?

Answer 16

structural analogues are structurally similar to the ligand and therefore can bind to the binding site.
structural analogues do not react with the protein in the way a ligand would.
structural analogues are usually non-hydrolysable.

Question 17

define non-hydrolysable.

Answer 17

non-hydrolysable means that the molecule is not susceptible to hydrolysis, meaning that the protein is unable to change the ligand as it normally would.

Question 18

give an example of a structural analogue.

Answer 18

• methotrexate > a structural analogue of dihydroflorate, which is the substrate for the enzyme dihydroflorate reductase.

Question 19

what is chemical modification?

Answer 19

chemical modification is a technique that selects particular amino acids and chemically modifies them, the amino acids selected are thought to be crucial to the protein/enzymes function.
chemical modification results in an alteration of the activity of the enzyme, therefore indicating the importance of the amino acid in that proteins binding site.
if the proteins activity is altered after modification, the amino acid modified is crucial in the binding site.

Question 20

give an example of chemical modification.

Answer 20

phosphorylation.

Question 21

what is pH dependence of activity?

Answer 21

altering pH affects acidic and basic residues leading to a shift in the equilibrium point of ionisation.
changing pH affects any amino acids with acidic or basic side chains, so this can potentially be used to investigate the roles of particular amino acid residues in enzyme activity.
a comparison of activity or of specificity of binding before and after the change in pH could reveal if the amino acid residue was in any way critical to the proteins function.