Lecture 11: HOW DOES AN ENZYME CATALYSE A REACTION?2 Flashcards
What does a progress curve do?
Measure the appearance of product or disappearance of substrate with time at a steady state
When is initial reaction velocity measured?
At/near time zero
What happens if there is sufficient excess of substrate?
As the amount of enzyme increases, the rate of reaction increases
What is initial velocity proportional to?
Enzyme concentration when substrate is in excess
How is data usually collected?
With a fixed amount of enzyme and variable amount of substrate
What happens to the initial rate as substrate concentration is increased?
At first when there is a low substrate concentration, it increases linearly and then as all the enzyme active sites become occupied, the rate of reaction stops increasing
What kinetic parameters can be identified on a velocity vs substrate concentration graph?
maximum velocity and Km
What is maximum velocity?
The maximum velocity possibly when substrate concentration is infinity
What is Km?
The substrate concentration at which velocity=maximum velocity/2. The Michaelis constant
What does a better enzyme have?
A smalle Km
What did enzymes evolve?
A Km which matched the substrate concentration in the cell
How I binding and catalyses described?
An enzyme, E, converts a single substrate, S, to a single product P that is instantly released.
What do we assume is irreversible?
Conversion of products back to the enzyme substrate complex
What do the relative speeds of K1 and K-1 do?
Define how tightly substrate binds
What is the rate of catalysis?
K2
What does K2 relate to?
Energy of activation for the transition state
What does the Michaelis-menten equation describe?
The V-obs vs substrate concentration curve
What does [ES]/[E] total describe?
The fraction of vmax the enzyme is running at
What are the assumptions to do with the Michaelis-menten equation?
Product is not converted back to substrate, Haldane’s steady state assumption, measuring initial rate insures [S] does not change significantly, [S] is much greater than [E] and all ES complexes have the same rate of reaction
What is Haldane’s steady state assumption?
The rate of ES formation equals the rate of its breakdown
What is necessary for the reaction?
The complex ES
What governs the rate of the reaction at any time?
[ES]
What does steady state refer to?
Time when [ES] does not change
What is [ES]+[E]?
Always constant
What kinetics does ES convert to E+P with?
First order
If each ES complex has the same chance of going through the transition state…
The ES»>E+P step will follow first order kinetics
What does cellular activity of enzyme activity often do?
Violate Michaelis menten
At any given time…
Some enzymes need to turn on and others need to drop out
What are the control mechanisms for enzymes?
Enzyme amount, allosteric control, cell location, proteolytic activation and post-translational modification
What do allosteric enzymes respond to?
Effectors binding away from the active site
What does binding accompany in allosteric enzymes?
A change of shape, which in turn changes enzymatic activity
What do allosteric enzymes often have?
Multiple subunits and display cooperative behaviour
What do co-operativity and allostery depend on?
The enzyme switching between active and inactive forms
Do cooperative enzymes follow Michaelis-menten equation?
No
What is the graph of v-obs vs [S] in cooperative enzymes?
Sigmoidal, not hyperbolic
What do cooperative enzyme respond to?
More steeply to intermediate changes in[S]
Where do cooperative enzymes evolve?
At regulatory points in metabolic pathways
What are some allosteric enzymes?
Aspartate transcrabamylase (ATCase) and phosphofructokinase
What does phosphofructokinase control?
Glycolysis
What is the action of phosphofructokinase?
It phosphorylates fructose-6-phosphate (F6P) to fructose biphosphate
When is phosphofructokinase inhibited?
If cell has plenty of ATP and glycolysis is not needed for energy
What type of protein is phosphofructokinase?
A homotetramer
When is phosphofructokinase cooperative?
When inhibited by ATP or phosphoenolpyruvate (PEP)
How does the T state of phosphofructokinase react?
Slowly
How does the R state of phosphofructokinase react?
Quickly
How is the T state in phosphofructokinase?
More compact, stabilised by PEP, an abundant intermediate of glycolysis
What is the R state of phosphofructokinase stabilised by?
Substrate FGP anf ADP
What changes when switching between T and R state of phosphofructokinase?
Arginine 162 and glutamic acid 161 swap positions in the active site
Where are zymogens secreted from?
The pancreas in inactive form
What does cleavage of zymogens by proteases in the gut do?
Produces active enzymes
What control are pancreatic digestive enzymes under?
Temporal and spatial
