Week 7 - Enzyme Function

Question 0

Scientists “would be interested to understand how fast the enzyme works and how much affinity the enzyme has for its substrate(s).”

Describe the [Substrate] against [velocity] curve produced

Answer 0

At low concentrations of substrate, V is directly proportional, but then graph curves asymptotically. This shows that at low conc. of substrate, the substrate is the limiting factor and enzyme quickly converts it to product after binding.

Whereas at high concentrations of substrate, enzyme is finding it hard to keep up.

Question 1

What is Vmax, and what is Kcat?

Answer 1

Vmax - maximum possible reaction velocity when all enzyme molecules exist as enzyme-substrate complexes

At Vmax, [enzyme] = [enzyme-substrate complex]

Kcat - independent of enzyme concentration unlike Vmax, it is the maximum number of times a single molecule of enzyme can perform its reaction in 1 second.. A Kcat of 1000 means each enzyme is catalysing 1000 molecules of product. Measured in time -1.

Vmax = Kcat X [enzyme]

Question 2

What is Km?

Answer 2

It’s a substrate concentration that reflects “the affinity an enzyme has for its substrate”
It is the amount of substrate needed for an enzyme to reach 1/2 Vmax

Question 3

How is Km related to affinity of an enzyme for its substrate?

Answer 3

Inversely related.
High Km means a low affinity for its subsrate.
Low Km means a high affinity for its substrate.

Question 4

What are the units of Km. Is it better to have a high or low Km?

Answer 4

Units are [concentration] as its a substrate concentration.

Better to have a low Km, reflecting enzyme has a high affinity for substrate. It also means it takes little substrate to get to 1/2 Vmax

Question 5

Perfect enzymes have a high specificity constant I.e. A high Kcat/Km. Explain why it is good to have a high specificity constant. Also mention why not all enzymes are perfect.

Answer 5

A high Kcat/Km means enzyme has a high turn over number and a high affinity for its substrate.

However not all enzymes are perfect because having a high velocity can be dangerous, enzymes become harder to control.

Question 6

What is the Michaelis-Menten equation?

Answer 6

V = Vmax X [S]
_________
Km + [S]

Describes the hyperbolic curve between [S] and V

Question 7

How do we determine Vmax, Km?

Answer 7

Lineweaver - Burke plot
- double reciprocal (see the maths behind how it’s derived in notes!)

1 = Km . 1 + 1
_. ___ _ _
V Vmax [S] Vmax

This corresponds to equation of straight line, y = mx + c

Y intercept corresponds to 1/Vmax
X intercept corresponds to -1/Km

Question 8

L-B plot can be useful for determining how an inhibitor binds. What are the changes in Vmax and Km in compete titmice and non competitive inhibition

Answer 8

1) competitive Inhibition
Inhibitor binds to enzyme active site, preventing substrate binding. This means that affinity for substrate decreases. At high substrate concentrations, the inhibitor doesn’t compete well so Vmax isn’t changed. So..

Km DECREASES
VMAX STAYS THE SAME

2) Non-competitive inhibition
Inhibitor doesn’t bing to active site, therefore affinity for substrate remains the same. It doesn’t matter if there is high substrate concentration, as a conformation change has occurred. So..

Km STAYS THE SAME
VMAX DECREASES

Question 9

What is the apparent Km and how does it change in terms of competitive inhibition?

Answer 9

Apparent Km is the Km in presence of competitive inhibitor.

In presence of competitive inhibitor;
more substrate is needed to counter the inhibitor and form enzyme- substrate complexes
More substrate is needed to get to the same enzyme velocity compared to no competitive inhibition
More substrate is required to get to 1/2 Vmax
Km therefore increases
Apparent affinity for an enzyme for substrate decreases

Km in presence comp. inhibitor = Km (apparent) = 
Km X (1 + [I] / Ki) 

I is inhibitor conc,
Ki is inhibitor constant (low Ki makes a good inhibitor)

Question 10

Ampicillin type antibiotics exert a different type of inhibition that is unlike the others, irreversible. Describe this type of inhibition

Answer 10

Inhibitor binds to active site of the enzyme and then COVALENTLY binds to active site of enzyme. This is irreversible inhibition and is terms suicide inhibition.

Kinetics would resemble that of non-competitive inhibition because you decrease the active enzyme amount.