11 - Enzymes VI: Activity and regulation Flashcards
Factors affecting enzyme activity
- Inhibitors
- Physical factors
- Cellular regulation
Physical factors: temperature
- As temperature increases, collisions between substrates and active sites occur more frequently as molecules move faster
- Thermal agitation disrupt the weak bonds that stabilise the protein conformation, leading to thermal denaturation
Physical factors: pH
- pH influences protein conformation and electrostatic interactions between enzyme and substrate (ionisation).
- Optimum between pH 6–8 for most enzymes
- However, digestive enzymes in the stomach are designed to work best at pH 2 while those in the intestine are optimal at pH 8, both matching their working environments
How much enzyme activity is present in a cell?
A. Two major factors:
1. How much enzyme is present
– A balance between rates of synthesis & degradation
2. The absolute activity of the enzyme present
– Regulation
changes in [S] and [P]
• [S] - more substrate means more product
• High [P] can inhibit enzyme activity - Product inhibition
BUT:
• [S] and [P] only affect overall measured rates of reaction and not the catalytic properties of an individual enzyme.
Feedback inhibition
• Biochemical pathways often have a committed step:
- The first dedicated reaction (e.g. only final product is F)
- Often the rate-limiting step
• Final product of pathway often behaves as an inhibitor of the enzyme that catalyses the committed step.
Feedback inhibition: 3-PGDH
3-phosphoglycerate dehydrogenase Tetramer of 4 identical subunits: • 4 catalytic sites • 4 regulatory serine-binding sites - lowers Vmax for 3-PG • Preserves 3-PG levels in the cell
Allosteric regulation
• Usually associated with multi-subunit enzymes
• There may be positive and negative regulators involved
– Positive: cooperative binding
– Negative: allosteric inhibitors
• Allows feedback control of metabolic pathways
– Input and output
Allosteric effect
an effect at one site on a molecule caused by binding of a molecule at a second, distinct site
Positive cooperativity
- Allosteric enzymes do not obey Michaelis-Menten kinetics
- Rate vs. [S] plot is sigmoidal
- Substrate binding at one site decreases the KM at other active sites
- Results in rapid responses to increases in [S]i.e. steeper curve
Sigmoidal plot
combination of two Michaelis-Menten type plots:
• T-state = “tense” - low activity
• R-state = “relaxed” - high activity
• Switch from T to R decreases KM & increases reaction rate
Aspartate carbamoyltransferase (ACTase)
- ACTase is the first committed step in pyrimidine synthesis
- End points are nucleotides, including cytidine triphosphate
- Classic example of allosteric regulation
- Activity stimulated by the substrate aspartate -Positive allosteric regulation
- Activity inhibited by CTP - Negative allosteric regulation
ACTase structure (multi-subunit)
• Catalytic subunit – Binds substrates – Catalyses formation of products – Not affected by CTP • Regulatory subunit – Binds CTP – No catalytic activity Complete structure has 6 catalytic & 6 regulatory subunits
ACTase structure (general)
Two layers of catalytic trimers surrounded by regulatory dimers
ACTase: reaction mechanism
• PALA is a stable transition state analogue- forms a stable complex for structure determination
