Enzyme Kinetics

Question 1

Define Enzyme Kinetics

Answer 1

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

Question 2

What is reaction rate?

Answer 2

The decrease in amount of substrate/ increase in amount of product formed per unit time

Question 3

Describe the Reaction rate vs Substrate concentration graph

Answer 3

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

Question 4

Describe the Michaelis Menten Reaction Model

Answer 4

k1 k2
E + S ES———–> E + P
k -1

Question 5

What are you assuming in the MM reaction model?

Answer 5

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

Question 6

What is Michaelis-Menten Equation?

Answer 6

V0 = (V max [S]) /( Km + [S])

Question 7

Define the initial velocity

Answer 7

the velocity when the enzyme and substrate are initially mixed

Question 8

Define the max velocity

Answer 8

The velocity of the enzyme catalysed reaction when all the active sites are fully saturated with substrate

Question 9

Define the Michaelis Constant

Answer 9

Km = (K-1 + K2) / K1

Question 10

What is Km equal to if V0 = 0.5 Vmax?

Answer 10

[S]

Question 11

Describe the effect of [S] on V0 for 2 enzymes

Answer 11

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

Question 12

What occurs when K2 is less than K1?

Answer 12

(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

Question 13

Define Kcat

Answer 13

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

Question 14

How would one compare Catalytic efficiency ?

Answer 14

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

Question 15

What is the Lineweaver-burke plot?

Answer 15

it is the recripcal function to the MM equation

Question 16

When can we extrapolate from the LB plot

Answer 16

when 1/[S] is negative - useful when we want to find out t the x intercept (-1/Km)

Question 17

What is lactate dehydrogenase?

Answer 17

catalyses the production of sugar to energy

Question 18

Where is 4M found and what properties does it exhibit

Answer 18

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

Question 19

Where is 4H found and what properties does it exhibit

Answer 19

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.

Question 20

What is the mode of competitive inhibitors

Answer 20

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

Question 21

What is the mode of non competitive inhibitors

Answer 21

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

Question 22

How is enzyme activity regulated

Answer 22

Allsoteric binding sites
Covalent modification
Induction/Repression of enzyme synthesis

Question 23

Describe allosteric regulation

Answer 23

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.

Question 24

Describe Covalent modification

Answer 24

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)

Question 25

Give an example of induction and repression of enzyme synthesis

Answer 25

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