Enzyme Kinetics Flashcards
Define Enzyme Kinetics
The study about the rate of an enzyme catalysed reaction and how it varies with different
substrate concentrations amounts of inhibitors metal ion concentration co factor concentration pH
What is reaction rate?
The decrease in amount of substrate/ increase in amount of product formed per unit time
Describe the Reaction rate vs Substrate concentration graph
At low substrate concentration- reaction rate is directly proportional to the the substrate concentration- 1st order reaction
At high substrate concentration- reaction rate is independent to the substrate concentration (the rate of increase of reaction rate decreases) - 0 order reaction
There is a max line which the curve never meets- it will never meet this line in reality as there can never be infinite amount of ES
Describe the Michaelis Menten Reaction Model
k1 k2
E + S ES———–> E + P
k -1
What are you assuming in the MM reaction model?
The [S] is greater than [E] so that at any time amount of substrate bound by enzyme is small
[ES] does not change due to the formation of ES = breakdown of ES ( ie the reaction is under steady state)
As initial velocities are used [P] is small so P—–> S is negligible and small
What is Michaelis-Menten Equation?
V0 = (V max [S]) /( Km + [S])
Define the initial velocity
the velocity when the enzyme and substrate are initially mixed
Define the max velocity
The velocity of the enzyme catalysed reaction when all the active sites are fully saturated with substrate
Define the Michaelis Constant
Km = (K-1 + K2) / K1
What is Km equal to if V0 = 0.5 Vmax?
[S]
Describe the effect of [S] on V0 for 2 enzymes
The Vmax for 2 enzymes would be the same as it is theoretical and there are no inhibitors
However the Michaelis constant would be different- So to achieve the same 0.5Vmax a different substrate concentration is required
What occurs when K2 is less than K1?
(when the affinity between Enzyme and Product is low)
K2 is rate limiting as a result - negligible-
Km = K -1 / K1 - This is known as the dissociation constant whereby Km will represent the affinity of the substrate with the active site
Define Kcat
It is a turnover number- the number of substrate molecules converted to product in a given unit time
on a single enzyme molecule
enzyme is saturated with substrate
How would one compare Catalytic efficiency ?
Kcatt/ Km since 2 enzymes may have the same Kcat but different Km. The higher this constant the better the enzyme. This is because the smaller the value of Km is the greater the affinity and the greater the Kcat value the greater the turnover value is
What is the Lineweaver-burke plot?
it is the recripcal function to the MM equation
When can we extrapolate from the LB plot
when 1/[S] is negative - useful when we want to find out t the x intercept (-1/Km)
What is lactate dehydrogenase?
catalyses the production of sugar to energy
Where is 4M found and what properties does it exhibit
Found in skeletal muscle- will have a high Kcat and high Km hence will ensure a big turnover in the lactate being produced in the cells
Where is 4H found and what properties does it exhibit
Found in heart muscle- will have a low Kcat and low Km hence will ensure the enzyme has higher affinity to substrate to help ensure it can sequester the substrate.
What is the mode of competitive inhibitors
Blocks the enzyme active site- by mimicking the substrate eg. malonate inhibiting succinate dehydrogenase
Competitive inhibitors alter the Km (increases it) which means that the affinity of substrate to active site is less. Greater [S] required to achieve the same 0.5 Vmax
with MM plot- the x intercept becomes less negative
Increasing the [S] will decrease the effect of the inhibitor so will increase the rate of reaction
Vmax stays the same- with the LB plot y intercept stays the same
What is the mode of non competitive inhibitors
They interfere in some other way with catalytic mechanism not with the active site- can either be reversible (inhibition of EDTA with Mg2+) or non reversible ( orgenphosphorius inhibition of cholinesterase)
Vmax with NCI is small than Vmax without it- the y intercept in LB plot will be Lower and the plateau in MM plot will be lower.
Km will remain the same as the [S] required to achieve 0.5 Vmax stays the same. In the LB plot the x intercept stays the same
How is enzyme activity regulated
Allsoteric binding sites
Covalent modification
Induction/Repression of enzyme synthesis
Describe allosteric regulation
The shape of the active site can be changed by positive or negative inhibitors
With positive effectors- the substrate can more easily fit in to the active site- phosphoenolpyruvate (PEP) and fructose 1,6 bis phosphate on pyruvate kinase.
With negative effectors- substrate detaches or does not fit into the active site- TP and citrate on phosphofructokinse.
Describe Covalent modification
Phosphate groups being added to Ser,Thr,Tyr, His
Phoshphroylation of glycogen phosphate increases its activity-done by phosphorylating serine 14 by phosphorylation kinase
dephosphoryaltion at serine 14 by phosphorylase phosphptase
Phosphorylation decreases the activity glycogen synthase (synthesises glycogen).
Phosphorylation at several serine residues (by glycogen synthase kinase 3) inactivates the enzyme. Dephosphorylation by a phosphoprotein phosphatase.
Adenylylation - Tyr residues (ATP to PPi)
• Uridylylation - Tyr residues (UTP to PPi)
• ADP-ribosylation - Arg, Gln, Cys residues (NAD to nicotinamide)
• Methylation - Glu residues (S-adenosyl-methionine to S-adenosyl-homocysteine)
Give an example of induction and repression of enzyme synthesis
igh blood glucose levels lead to an increase in insulin production. Insulin increases rate of synthesis of key enzymes involved in glucose metabolism:
• Glucokinase
• Phosphofructokinase
• Pyruvate kinase
