Regulation, Enzymes and Rate Limiting Steps Flashcards
Describe the chemical features of ATP which make it ideal for use as an energy currency
The bonds between the phosphate groups are unstable and rich in energy, making them readily cleavable. When these bonds are broken, energy is released, providing a source of readily available and quickly mobilizable energy–> This hydrolysis is exergonic (energy-releasing), providing energy for cellular processes.
ATP is a water-soluble molecule due to its numerous polar functional groups, including phosphate groups
ATP is readily available within cells upon demand as stores are released in manageable amounts, meaning there is no wasted energy. [ Pool of compounds able to do substrate level phosphorylation]
ATP serves as a immediate energy storage molecule.
ATP can be regenerated in cellular processes like cellular respiration and photosynthesis = continous supply
Explain the concept of energy charge with reference to the concentration of adenine nucleotides
“energy charge” to the ratio of high-energy phosphate compounds (ATP), to lower-energy compounds (ADP) and (AMP)
The energy charge close to 1 (maximum energy charge)
= high energy charge indicates a cell with sufficient ATP/Lower energy charge suggests a cell with a higher demand for energy.
Key Points:
- Large, abrupt changes in ATP concentrations are not desirable = WANT relatively constant level of ATP to ensure stability in energy availability.
- Keeping ATP at 5 mM: target concentration for maintaining cellular energy levels = sufficient for the immediate energy needs of the cell.
- The concept recognizes the need for instant reserves of energy. The pool of compounds capable of substrate-level phosphorylation, such as creatine phosphate, serves as a rapid source of ATP production but can only sustain energy supply for a few seconds.
- Increasing Catabolic Pathways: To replenish ATP levels and meet the cell’s energy demands= release energy and regenerate ATP.
Review how a small change in ATP concentration is translated into a large relative change in AMP concentration
since ATP > ADP»_space; AMP
The decrease in ATP results in a more substantial relative increase in the concentrations of ADP and AMP.
The reason for the sensitivity lies in the fact that ATP and ADP concentrations are typically much higher than AMP. Therefore, a small change in ATP has a more pronounced effect on the relative increase in AMP.
Interpret enzyme kinetic parameters to identify potential rate limiting steps
Km (Michaelis-Menten Constant): the substrate concentration at which the enzyme works at half of its maximum velocity (Vmax).
A high Km indicates that the enzyme has a lower affinity for its substrate. This suggests that the enzyme is less efficient at low substrate concentrations.
A low Km indicates a higher affinity for the substrate, meaning the enzyme is efficient even at low substrate concentrations.
Vmax (Maximum Velocity): Vmax is the maximum rate of the reaction, representing the enzyme’s efficiency when all active sites are saturated with substrate.
A low Vmax suggests that the enzyme has a lower overall catalytic efficiency, potentially due to limitations imposed by the rate-limiting step.
A high Vmax indicates a more efficient enzyme, capable of catalyzing the reaction at a higher rate.
Turnover Number (kcat): the number of substrate molecules converted to product per enzyme active site per unit time.
A low kcat suggests that the enzyme is less efficient in converting substrate to product, possibly due to limitations imposed by the rate-limiting step.
A high kcat indicates a more efficient enzyme, capable of catalyzing the reaction at a higher rate.
Substrate Saturation:
If the enzyme is saturated with substrate (i.e., working at Vmax) at physiological substrate concentrations, it suggests that the reaction may be limited by downstream steps in the pathway.
Describe the properties of rate limiting steps
Properties of Rate-Limiting Steps (RLS):
* Irreversible: The RLS is often an irreversible step, meaning once it occurs, it cannot easily be reversed.
* Alternative Enzymes: Since the RLS is irreversible, alternative enzymes are needed if the pathway needs to be reversed or if a different product is desired.
* Not at Equilibrium: The RLS is not at equilibrium under physiological conditions, as it directs the pathway toward the production of specific products.
* Committed Steps: The RLS is often referred to as a committed step because it commits the substrate to continue through the pathway.
* Saturated with Substrate: The RLS is saturated with substrate, meaning the active sites of the enzyme are fully occupied.
* Low Km or [S]»_space; Km: The RLS typically has a low Km value, or the substrate concentration at the RLS is much higher than the Km value.
* Working at Vmax: The RLS operates at Vmax, as it is the slowest step and determines the maximum rate of the pathway.
Review the major ways in which enzyme activity can be changed
a. Make the Rate-Limiting Enzyme Go Faster/Slower:
“Make ticket-reading & gate-opening happen faster.”
Directly modulating the activity of the enzyme responsible for the rate-limiting step can control the overall flux through the pathway.
b. Turn the Rate-Limiting Enzyme On/Off or Make It Work the Other Way:
“Switch them from being ‘off’ to ‘on’ or change the direction from ‘in’ to ‘out.’”
This refers to controlling the activation or inhibition of the rate-limiting enzyme. Turning it on or off, or changing its direction, can significantly impact the pathway’s output.
c. Increase the Rate of Transcription/Translation of the RLS – or Change Its Rate of Degradation:
“Bring in a set of gates when you need them.”
Modulating the synthesis or breakdown of the rate-limiting enzyme at the genetic or post-translational level can effectively control its abundance and, consequently, the pathway’s activity.
List the key rate limiting steps in the major pathways of catabolism
fatty acid oxidation
availabilty of CoA might be the rate limiting pathway : trap FA in cytoplasm to form FA-CoA
availability of carinitie might be the rate limiting pathway :
dehydrogenase (break fatty acid chain into 2 carbon chunks of acetyl CoA) is regulated
CoA, NAD, FAD = could be rate limiting factors in fatty acid oxidation
glucose oxidation
GLUTS to transport glucose
HK/PFK/PK = enzyme activity
we can regulate glycogen phosphorylase to convert glycogen to G6P
hexokinase = 1st enzyme in glycolysis, adds phosphate to glucose to make G6P
phosphofructokinase
pyruvate kinase regulated in glycolysis
PDH to convert pyruvate to acetyl CoA
no NADH in glycolysis
few key enzymes are regulated, availability of NAD and transporters
Provide an overview of the regulation of phosphofructokinase
PFK controls the conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6BP).
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Allosteric Inhibition by ATP:
- PFK is allosterically inhibited by high concentrations of ATP.
- Mechanism: ATP binds to an allosteric site on PFK, causing a conformational change that reduces the enzyme’s affinity for its substrate, F6P.
- Effect: This inhibition prevents unnecessary ATP consumption when cellular energy levels are already high.
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Allosteric Activation by AMP:
- PFK has an allosteric site for AMP, and binding of AMP influences the enzyme’s response to ATP.
- Mechanism: AMP binding induces a conformational change in PFK, making it less sensitive to ATP inhibition.
- Effect: When cellular energy levels are low (indicated by a high AMP/ATP ratio), AMP binding activates PFK, promoting glycolytic flux.
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Allosteric Inhibition by Citrate:
- Citrate, an intermediate of the citric acid (TCA) cycle, allosterically inhibits PFK.
- Mechanism: Citrate binding to PFK negatively modulates its activity.
- Effect: This inhibition occurs when there is an excess of citrate, suggesting that the TCA cycle is well-fed, and glycolysis can be slowed down to prevent an excessive accumulation of glycolytic intermediates.
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Regulation by Fructose-2,6-Bisphosphate (F2,6BP):
- An additional level of regulation involves F2,6BP, which activates PFK.
- Mechanism: F2,6BP is a powerful activator that binds to a regulatory site on PFK, stimulating glycolytic flux.
- Effect: This activation occurs when glucose levels are high and glycolysis needs to be accelerated.
Provide an overview of the regulation of hexokinase
Hexokinase : phosphorylation of glucose –> (G6P) in the first step of glycolysis.
1) Feedback Inhibition by G6P:
binding of G6P induces a conformational change in hexokinase, reducing its catalytic activity.
Effect: This feedback inhibition prevents the excessive phosphorylation of glucose when G6P levels are already high, ensuring that glucose is not continuously trapped within the cell.
2) Allowing Glucose Efflux:
Hexokinase activity traps glucose within the cell by converting it to G6P.
glucose is not phosphorylated when hexokinase is inhibited, allowing it to diffuse back out of the cell.
3) Prevention of ATP Waste:
Hexokinase phosphorylates glucose using ATP as a substrate.
Energy Conservation: Feedback inhibition prevents the unnecessary consumption of ATP when G6P levels are sufficient. This ensures that ATP is not wasted on phosphorylating glucose when it is not needed for cellular metabolism.
Equivalent Feedback Inhibition at the Gates:
–> role in controlling the entry of glucose into glycolysis and maintaining cellular energy balance.
Using an example, illustrate how control motifs act synergistically to regulate pathways
PFK : converts fructose 6-phosphate to fructose 1,6-bisphosphate
PK : pyruvate kinase
–> When cellular ATP levels are sufficient (ATP concentration is high), ATP binds allostericaly to PFK-1 and PK, inhibiting its activity and slowing down glycolysis.
Feedforward Activation: As glycolysis progresses and fructose-1,6-bisphosphate increases, it binds to PK and further enchances its enzyme activity
when concentration of AMP increases (insufficient energy) = no hexokinase inhibition anymore
activates PFK and PK –> switch on glycolysis
Using an example, show how enzymes are controlled by reversible phosphorylation
PDH : pyruvate dehydrogenase converts pyruvate to acetyl CoA
PDH is inactive when bound to phosphate
Dephosphorylation:
PDH phosphatase removes phosphate group = active [insulin : for the use of glucose for energy, energy demand is high]
Phosphorylation:
PDH kinase adds phosphate group = inactive [high levels of acetyl CoA and energy demand is low]
acetyl CoA binds to PDH kinase to switch on inactivation of PDH by phosphorylation
