Lecture 8

Question 1

What does Wilhelmy show in 1850?

Answer 1

Rate of acid hydrolysis of sucrose is proportional to sucrose concentration at constant acid concentration.

Question 2

In 1902, what does Brown find?

Answer 2

There are different kinetics for the same reaction catalysed by invertase. At low sucrose concentrations, rate proportional to sucrose concentration, but independent at high sucrose concentrations.

Question 3

Enzymes become saturated by substrate at higher substrate concentrations. What is the only way to increase reaction rates? Furthermore, what is this used as evidence for?

Answer 3

Add more enzyme. Evidence for formation of discrete enzyme substrate complex intermediate between substrate and products.

Question 4

What did Leonor Michaelis and Maud Menten do and when?

Answer 4

Provided mathematical solution describing rectangular hyperbolic nature of rate vs [substrate] plot. 1913.

Question 5

What is the Michaelis Menten equation?

Answer 5

Question 6

What is K_M?

What does it mean?

Answer 6

The Michaelis constant.

Means the concentration of Substrate ([S]) at half V_max.

Question 7

What is V_max?

Answer 7

Maximum rate of reaction.

Question 8

What is the equation showing the equilibrium between Substrate and Product?

Answer 8

Question 9

What equation forms the original basis of which the Michaelis Menten equation was developed?

Answer 9

Question 10

What is assumed about k2?

Answer 10

It is a negligible rate, therefore happening in the forward direction only.

Question 11

What is the unit used for K_M?

Answer 11

M.

Question 12

What does K_M represent in terms of the amount of enzyme occupied?

Answer 12

Represents the substrate concentration at which the rate (and therefore the number of active sites occupied) is half maximal.

Question 13

What does K_M often approximate the affinity of?

Answer 13

The enzyme to the substrate.

Question 14

What does a low value for K_M mean for the affinity of the substrate?

Answer 14

High affinity, i.e. strong/tight binding.

Question 15

In many cases, what is the in vivo substrate concentration close to?

Answer 15

The value for K_M.

Question 16

Why is K_M a constant?

Answer 16

Because it defines the interaction between enzyme and substrate molecules.

Question 17

What is K_M independent of?

Answer 17

Amount of enzyme and substrate present.

Question 18

Is V_max a constant? Why?

Answer 18

No.

Measured value only applies to one particular reaction condition.

Question 19

Will adding more enzyme increase K_M and V_max?

Answer 19

Will increase Vmax but not K_M.

Question 20

When is V_max reached? And can this be done in practice?

Answer 20

When all substrate binding sites are full.

No, only theoretical.

Question 21

What is the turnover number?

And what’s the units?

Answer 21

Number of substrate molecules converted into product per unit time when the enzyme is fully saturated with substrate.

s^-1.

Question 22

What does the turnover number mean in real terms?

Answer 22

The efficiency of the enzyme.

Question 23

What symbol can be used to describe the turnover number?

Answer 23

k_cat.

Question 24

What 2 things is k_cat equal to?

Answer 24

Kinetic constant k₂.

V_max/amount of enzyme.

Question 25

How can V_max be calculated from k₂?

Answer 25

k₂ x amount of enzyme.

Question 26

2 steps to estimate K_M and V_max?

Answer 26

Perform enzyme assays under identical conditions at range of substrate concentrations. Plot initial rate, V₀ against [S]

Question 27

What type of plot is the Lineweaver-Burk plot?

Answer 27

Double reciprocal plot.

Question 28

What does the slope of the Lineweaver-Burk plot represent?

Answer 28

K_M/V_max.

Question 29

What does the x-intercept represent on a Lineweaver-Burk plot?

Answer 29

-1/K_M

Question 30

What does the y-intercept represent on a Lineweaver-Burk plot?

Answer 30

1/V_max.

Question 31

What type of plot is best to convert protein-ligand binding curve to a straight line, but is not often used?

Answer 31

Eadie-Hoftsee plot.

Question 32

What does the y-intercept represent on the Eadie-Hoftsee plot?

Answer 32

V_max.

Question 33

What does the x-intercept represent on the Eadie-Hoftsee plot?

Answer 33

V_max/K_M.

Question 34

What does the gradient represent on the Eadie-Hoftsee plot?

Answer 34

-K_M.

Question 35

Why do V_max and K_M matter? (3 reasons)

Answer 35

* Helps understand flux through biochemical pathways. * K_M can provide rough estimate of substrate concentration in cell. * Identification and design of drugs.