Enzyme Kinetics

Question 1

Describe the protein chymotrypsin?

Answer 1

241 amino acids, 3 peptide chains A, B, C linked by disulphide bridges.

Serine protease

Secreted by the pancreas as pro-enzyme chymotrypsinogen

Does proteolysis in duodenum by hydrolysing peptide bonds

Needs aromatic side chains e.g. phenylalanine, tyrosine, tryptophan

Question 2

What does chymotrypsin catalyse GPNA into?

Answer 2

N-Glutaryl L-Phenyl alanine and p-nitroaniline

Question 3

What is the beer-lambert law?

Answer 3

..

Question 4

What is the KM ( Michaelis constant ) ?

Answer 4

Concentration of substrate at which a particular enzyme works at half its maximum velocity

• useful for comparing the strength of enzyme-substrate complexes

Question 5

What does a Low KM indicate?

Answer 5

Tight binding of a substrate to an enzyme

• so high KM is indicative of weak binding

Question 6

How to calculate Vmax from equation?

Answer 6

Kcat * [Enzyme]

Question 7

How to calculate V0 ( initial velocity ) ?

Answer 7

Vmax [S] / (Km + [S]

Question 8

During the initial phase of the reaction, as long as the velocity remains constant, the reaction is in (x) =, with ES being formed and consumed (y)

Answer 8

x - steady state

y - at the same rate

Question 9

Describe a Lineweaver-Burk plot:

The axis
The intercepts
The gradient

Answer 9

X axis : 1/[S]

Y axis : 1/V0

Intercept at X axis : -1/KM

Gradient : KM/Vmax

Y intercept : 1/Vmax

Question 10

The low the Km value, (x) the affinity of the enzyme for its substrate ?

Answer 10

Higher

Question 11

Describe a lineweaver burk plot for a competitive inhibitor vs non-competitive inhibitor vs no inhibitor?

Answer 11

Competitive inhibitor has a different X axis intercept ( -1/KM value ) to no inhibitor whilst non-competitive has the same intercept.

Competitive inhibitor has the same Y axis intercept ( Vmax ) as no inhibitor whilst non-competitive has a different intercept ( higher )

Question 12

How can competitive inhibitors be overcome?

Answer 12

Putting in more substrate to outcompete the inhibitor = inhibits Km but nothing to Vmax

Question 13

What is the Kcat?

Answer 13

Turnover number : number of molecules that an enzyme can process in a given unit of time.

Question 14

How is Chymotrypsin actively inhibited?

Answer 14

By Indole - a molecule with a competitive active site. This increase Km value - lowering affinity whilst Vmax remains the same