Enzymes Flashcards
How do enzymes alter the rate of chemical reactions?
Catalysts that increase reaction rate by accelerating attainment of equilibrium. Lower Ea by optimising conditions energy-wise for the reaction.
Do not alter equilibrium of a chemical reaction
What are the 5 key features of active sites?
Occupies a small part of the enzyme (129 AAs approx)
Formed by AAs from different parts of the primary sequence
Crevices/clefts
Complementary to substrate
Substrates bind to enzymes by multiple weak bonds
How do you measure the rate of enzyme activity?
- Add a known concentration of enzyme and substrate
- Wait for an equilibrium to be reached
- Measure the rate of reaction and plot a graph of product against time, find initial velocity (gradient)
- Repeat and plot initial velocities for different substrate concs
What is the Michaelis-Menten equation and what is it used for?
Vo = (Vmax x substrate conc)/(Km + substrate conc)
Tells the affinity of the enzyme for the substrate
What is the significance of the Km value? What does a low Km mean?
Substrate conc at which Vmax /2
The lower the Km, the higher the affinity of the enzyme for the substrate
When measuring the rate of enzyme activity, what is the definition of 1 unit?
The amount of enzyme that produces 1 millimole of product per min under standard conditions
ie. Per litre (L) of serum or per gram (g) of tissue
What is the equation for the Lineweaver-Burk plot? What does it represent?
1/Vo = (Km/Vmax) x (1/substrate conc) + (1/Vmax)
Michaelis-Menten equation linearised
How do competitive inhibitors affect Km and Vmax values?
Km increases, Vmax unaffected
How do non-competitive inhibitors affect Km and Vmax?
Km unaffected, Vmax decreases
How do irreversible inhibitors work? How can this be overcome?
Form a covalent bond with the enzyme, chemically modified enzyme is “dead.” Can only be overcome by synthesis of new enzyme
What is the effect of irreversible inhibitors on Km and Vmax?
Km unaffected, Vmax decreases
Name 5 types of short term regulatory mechanisms for enzymes?
- Different enzyme forms (isoenzymes)
- Change in enzyme conformation (allosteric regulation)
- Reversible covalent modification (phosphorylation)
- Proteolytic activation
- Controlling the amount of enzyme present (gene expression)
What is an isoenzyme? How do they work?
Enzymes that catalyse the same reaction but have different amino acid sequences
Hence different activity and regulatory properties
How does allosteric regulation work?
Usually multi-subunit enzymes and exist in either T state (low affinity) or R state (high affinity)
Do not obey MM kinetics
Activators increase proportion of enzyme in R state and inhibitors increase the proportion of enzyme in T state
Give 2 examples of activators and 3 examples of inhibitors in allosteric regulation
Activators: AMP, fructose 2,6-bisphosphate
Inhibitors: Citrate, ATP, H+
Explain how these effectors work and the metabolic basis for why they work
Describe the roles of protein kinases and phosphatases in the phosphorylation of enzymes
Kinase: transfer terminal phosphate of ATP to the -OH group of Ser, Thr and Tyr
Phosphatase: cause hydrolysis removal of phosphoryl groups from proteins
Why is protein phosphorylation so effective?
The free energy of phosphorylation is large
Adds 2 negative charges
Phosphoryl groups can make H-bonds
Rate of phosphorylation/ dephosphorylation can be adjusted
Links energy status of cell to metabolism through ATP
Allows for amplification effects
Describe an enzyme cascade
When enzymes activate enzymes, the number of affected molecules increases in an enzyme cascade
What does proteolytic activation entail?
Inactive precursor molecules (zymogens) becoming activated and breaking peptide bonds irreversibly to regulate enzyme activity
Important when processes need to be tightly regulated
How does cleaving a protein activate a zymogen?
N-terminus peptide is cleaved, change in primary sequence plus rotation of newly formed N-terminus towards active site induces conformational change creating a functional active site
Give 3 examples of zymogens
Trypsinogen (to trypsin)
Proelastase (to elastase)
Prolipase (to lipase)
