enzymes mw%% + Flashcards
Active site complementary to substrate = no reaction!
Explain the allosteric control of enzymes?
- Allosteric effectors are cell metabolites, bind non-covalently to a site on the enzyme that is not the active site
- This changes the enzymes structure
- Some effectors are activators and some are inhibitors
- Allosteric effectors are examples of non-competitive inhibitors
Explain Covalent Modification?
•Many ways enzymes (and other proteins) can be regulated through reversible covalent modification e.g phosphorylation.
What is an Enzyme?
- Globular protein
- Biological catalyst that differs from a chemical catalyst
–Catalyses very high reaction rates
–Shows great reaction specificity
–Work in mild temperature/pH conditions
–Can be regulated
Enzyme Definitions?
- Apoenzyme–The protein component of an enzyme that contains a cofactor
- Holoenzyme –“whole enzyme” – the apoenzyme plus the cofactor(s)
why don’t Allosteric enzymes not follow M-M kinetics?
- Allosteric enzymes are made up of many subunits, which contain many active sites.
- One substrate binding to an enzyme subunit can cause changes in other active sites on other subunits
- This can lead to the concept of “cooperative binding” of substrate molecules
what is a coenzyme?
- Complex organic molecule, usually produced from a vitamin e.g NAD
What do Enzymes do?
They do NOT:
–Move reaction equilibria
–Make a non-spontaneous reaction spontaneous
They DO:
–Increase rates of spontaneous reactions
–Lower the activation energy of biochemical reactions
–Accelerate movement towards reaction equilibrium
What will affect an enzyme?
Temperature
- ↑ temp., ↑ molecule collisions
- ↑ temp., ↑ internal energy of molecules
- ↑ temp. will eventually denature enzyme
pH
- pH changes the charge of amino acids
- If the active site amino acids charge changes the enzyme will cease to function correctly
Inhibitors
Reaction Coordinate Diagram?
- Spontaneous reaction isn’t instantaneous because of the energy barrier
- Transition state shows the moment that chemical bonds are formed and broken
- Reaction could then go either way (revert back to S or change to P)
what is a free energy?
- Energy can be converted from one form to another but is neither created or destroyed.
- Useful” energy generated from cellular reactions is termed Gibbs Free-Energy (G).
- Spontaneous reactions must have a –ve ΔG value
how do irreversible inhibitors affect enzymes?
inhibitor binds to the enzyme in a covalent, and therefore irreversible way.
what is a Prosthetic group?
- Cofactor covalently bound to the enzyme or very tightly associated with the enzyme
Measuring enzyme activity
•In clinical samples, normal activity is often given an arbitrary value:
1 U/ml or 100% = normal
•Separate different forms of enzymes by electrophoresis and examine pattern
Enzymes and two or more substrates?
- Catalysing a reaction with two or more substrates usually involves transfer of groups from one substrate to the other
- This can occur in several ways:
–Random order or Ordered with a ternary complex (complex formed between two substrate molecules and an enzyme)
–No ternary complex formation
How can you measure enzyme kinetics?
- At low [S] you get an almost linear increase in V0 as ↑[S]
- At higher [S] the V0 changes very little in response to ↑[S]
- When [S] becomes so large that V0 changes are vanishingly small you get maximum reaction velocity, Vmax
- Vmax occurs because all of the enzyme active sites are saturated with substrate
how do non-competitive inhibitors affect enzymes?
- bind to enzymes non-covalently
- The substrate is still able to bind the active site, so Km of the substrate-enzyme complex remains unchanged
- Increasing substrate concentration does not change the inhibition, Vmax will decrease
- non-competitive inhibitors= unchanged Km values but the Vmax decreases.
how do Competitive inhibitors affect enzymes?
- bind to enzymes non-covalently ,resemble the substrate molecule,compete with the active site
- leads to a decrease in the affinity between the active site and the substrate, so the Km increases
- Increasing substrate concentration can overcome this inhibition, so the same Vmax can be achieved
- competitive inhibitors exhibit increased Km values, Vmax remains unchanged
what are the functions of Glucokinase and hexokinase?
They both catalyse:
- Glucose + ATP ⇒ Glucose-6-phosphate + ADP.
- When [blood glucose] goes UP, the glucokinase activity increases but hexokinase activity does not respond as it is already working at its Vmax
- When [blood glucose] is LOW, gluconeogensis releases glucose from the liver,
how does feedback inhibition occur?
- Some biochemical pathways regulate the enzymes involved through feedback inhibition.
- A build up of the end product of a pathway, or a key junction in a pathway, can ultimately slow the entire pathway
What can Km and Vmax tell us?
- Km gives you a clue to the affinity of the enzyme with it’s substrate.
- larger Km values indicate a less stable ES complex. Reduced affinity
- smaller Km values indicate a more stable ES complex. Higher affinity
- Vmax tells you how fast a reaction is proceeding when the enzyme is saturated with substrate
what is a cofactor?
- Non-protein component needed for activity
Active site complementary to transition state = CATALYSIS
Enzyme Reaction Coordinate Diagram?
- An enzyme will facilitate the change of S to P through intermediates.
- Enzymes form non-covalent bonds with substrate molecules, called the “binding energy” allowing them to take the reaction through a different path of reaction intermediates
