CW5-6: Enzymes Flashcards
What makes up the ‘Y’ shape of an antibody such as IGG?
- 2x Fab domain (containing antigen-binding sites)
- Contains a light chain and a heavy chain
- 1x Fc domain
- Contains a heavy chain
How can myosin be studied?
It can be cut into four strands.
- Trypsin can be used to cut the myosin into heavy meromyosin (HMM) and light meromyosin (LMM)
- Papain then cleaves off the head section, resultin in two parts

Define ‘heptad’.
A repeated sequence of seven amino acids.
How many amino acids form one full turn of an alpha helix?
3.6
If a right-handed alpha-helical coiled-coil is a heptad in length, how many turns is it?
2
How does carboxypeptidase hydrolyse other proteins?
What prevents it attacking itself?
It attacks them at their C-terminus.
Its C-terminus is hidden within the folds of the protein.
What is an enzyme?
A protein which catalyses biological reactions
What are the properties of an enzyme?
- Highly specific (only some substrates can fit into the active site)
- Unchanged after reaction (regenerated at the end)
- Can accelerate reactions up to 106-fold
- Cannot alter reaction equilibria (will end up at teh same point in both the presence and absence of catalyst, energetically speaking)
What’s the general equation for the conversion of substrate to product?
S ⇌ P
What are the two factors that determine the appearance of a product?
- Thermodynamic factors (energy changes)
- Kinetic factors (rate changes)
What must the value of ΔG be for a reaction to occur?
Negative
What effect does a catalyst have on activation energy (EA)?
A catalyst lowers the EA required, so the reaction occurs more easily.
What is the shape of the graph of substrate concentration vs. rate?
Rectangular hyperbola
How is KM important in relation to glucose?
Hexokinase has a KM of 0.1 mM, so when the concentration of glucose is low, more glucose is converted to glucose-6-phosphate, resulting in energy release. A build-up of G6P results in inhibition of hexokinase.
Glucokinase (found only in the liver) has a KM of 5 mM, so when the level of glucose reaches this level, G6P is formed and stored as glycogen in the liver.
What is the ratio Kcat:KM a good measure of?
Enzyme efficiency
An efficient enzyme has a high ratio (either high Kcat or low KM)
What is the active site of an enzyme?
A region where reactions occur. In general, active sites form only a small section of an enzyme. They create a microenvironment, e.g. they often exclude water.
What are the two theories of enzyme-substrate complex formation?
- Lock and key (Emil Fischer, 1890)
- Induced fit (Dan Koshland, 1958)
What are the issues with the lock and key theory of enzyme-substrate complex formation?
- There is no reason to suggest products want to be formed because they fit together snugly so there is no explanation for the equation:
ES → E + P1 + P2
- Why would the binding of a non-competitive inhibitor away from the active site prevent the enzyme doing its job?
What did Dan Koshland propose was a better model for enzyme-substrate complexes?
The induced fit model, where the intermediate is presented as a transition state conformation. This is under tension so it is more likley to form products.
This theory fits better with the data, and non-competitive inhibitors can now be rationalised and their activity explained.
What are the two main types of enzyme inhibitor? Describe both.
Give the two subgroups of one type of inhibitor.
- Irreversible – bind very tightly and generally form covalent bonds
-
Reversible – non-covalent
- Competitive – binds at the active site
- Non-competitive – binds at another site on the enzyme
How do nerve gases such as sarin work?
They are irreversible enzyme inhibitors. Sarin is an acetylcholinesterase inhibitor, so nervous signals do not stop and muscle contraction is permanent.
What effect do competitive inhibitors have on Vmax and KM?
V<strong>max</strong> unchanged because a high enough substrate concentration can overcome the inhibitor
KM increases because a higher substrate concentration is required to reach 0.5Vmax
What effect do non-competitive inhibitors have on Vmax and KM?
Vmax decreases because adding more substrate won’t overcome the effect of the inhibitor becuase the inhibitor binds to another part of the enzyme
KM will remain the same (see M-M graph)
Describe ribonucelase.
- 124 amino acids (13.7 kDa)
- Hydrolyses phosphodieaster bonds in RNA
- Releases free 3’-phosphate and 5’hydroxyl termini
- Bell-shaped pH curve with optimum around pH 7
- Tighter curve than proteins in general
- Catalysis by a pair of histidine residues
Describe the process of the alkali-catalysed hydrolysis of RNA.
- The deprotonated 2’ OH of the ribose acts as a nucleophile and attacks the adjacent phosphorus in the phosphodiester bond of the sugar-phosphate backbone of the RNA.
- There is a transition state, where the phosphorus is bonded to five oxygen atoms.
- The phosphorus then detaches from the oxygen connecting it to the adjacent sugar, resulting in ester cleavage of the RNA backbone.
- This produces a 2’,3’-cyclic phosphate that can then yield either a 2’- or a 3’-nucleotide when hydrolysed.
Which amino acids are involved in the mechanism of ribonuclease? Which is protonated and which is unprotonated?
His 12 – unprotonated so accepts proton
His 119 – protonated so donates proton
What is the evidence for the mechanism of ribonucleases?
- pH-dependence of reaction (tight bell curve)
- Use of small molecule inhibitor, e.g. iodoacetate
- Use of a non-hydrolysable substrate analog, e.g. altered ribonuclease
- Site-directed mutagenesis (null reaction uses alanine instead because it’s a typical non-reactive amino acid)
Why is the bell curve for the pH dependence of ribonuclease activity so tight?
The pK of histidine is around 6.5, so it is readily protonated/unprotonated
Small pH changes result in both histidine residues being protonated or unprotonated so there is no reaction
How does iodoacetate carboxymethylate histidine?
It donates an acetate (CH2COO–) to one of the histidines
What are substrate analogs? Give an example.
Chemical compounds with a chemical structure that resembles the substrate molecule in an enzyme-catalyzed chemical reaction. They can act as competitive inhibitors of an enzymatic reaction.
e.g. phosphonate is a substrate analog for phosphate ester. The enzyme thinks it’s the right substrate but cannot let it go because it cannot be processed by the enzyme due to the CH2 group (where the O would normally be)
