Enzyme kinetics

Question 1

Why does the reaction rate plateau after a while?

Answer 1

Substrate is getting depleted

Question 2

What are 3 ways to measure reaction rates?

Answer 2

Spectrophotometry, fluorescence, radioactivity

Question 3

How do you measure reaction rate with spectrophotometry?

Answer 3

Look for the appearance or disappearance of a coloured product, or if the substrate or product has a unique absorption spectrum

Question 4

Why is spectrophotometry a good way to measure the rate of redox reactions?

Answer 4

NADH absorbs UV light at 340 nm, and NAD+ doesn’t

Question 5

How do you measure reaction rate with fluorescence?

Answer 5

Look for the unique fluorescence emission spectrum of the substrate or product

Question 6

How do you measure reaction rate with radioactivity?

Answer 6

Look for the gain or loss of a radioactive group by taking samples at different time points and measuring radioactivity

Question 7

What type of graph do we get with the MM equation?

Answer 7

Hyperbolic, Vo vs [S]

Question 8

What is Vmax? Is it constant? What are the units?

Answer 8

The maximum reaction rate when the enzyme is completely saturated. It stays constant for a certain enzyme concentration.. Units are concentration/time

Question 9

What is the MM equation simplified to when [S]&raquo_space; Km?

Answer 9

Vo = Vmax

Question 10

What is the MM equation simplified to when [S] &laquo_space;Km?

Answer 10

Vo = Vmax [S]/Km. Which is a linear relationship

Question 11

What is the MM equation simplified to when [S] = Km?

Answer 11

Vo = Vmax/2

Question 12

What is kcat? Is it constant? What are the units?

Answer 12

The turnover number, which is the number of reactions per unit time. It stays constant for an enzyme regardless of concentration. Units are 1/time

Question 13

How do we calculate the time for one reaction?

Answer 13

1/kcat

Question 14

What is Km? Is it constant? What are the units?

Answer 14

The rate constant, the [S] at Vmax/2. Stays constant for a set of conditions

Question 15

When can we say that Km is a measurement of enzyme-substrate affinity?

Answer 15

When the enzyme follows a simple MM mechanism, where the actual reaction (ES -> E + P) is very slow. Km = Kd

Question 16

What is the best way to compare the reaction rates of enzymes?

Answer 16

The specificity constant, kcat/Km

Question 17

What is catalytic perfection? How do we know when an enzyme has achieved catalytic perfection?

Answer 17

The rate limiting step is the diffusion of the substrate into the active site, not the catalysis itself. Catalytic perfection is achieved when kcat/Km is between 10^8 and 10^9