PRACTICAL D: Enzyme kinetics

Question 1

What is pH optimum?

Answer 1

pH at which enzyme activity is maximised

Question 2

What is Km?

Answer 2

[S] at which the enzyme is half saturated and Vo is 1/2*Vmax

Question 3

What is Vmax?

Answer 3

The maximal velocity, or Vmax, is the rate of the reaction when there are enough substrate molecules to completely fill (saturate) the enzyme’s active sites.

Reminder: Increasing the substrate concentration indefinitely does not increase the rate of an enzyme-catalyzed reaction beyond a certain point.

Question 4

What is the activity of the enzyme?

Answer 4

mol.unit time-1

Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. A practical and commonly used value is 1 enzyme unit (U) = 1 μmol.min-1.

Question 5

What is the specific activity of the enzyme?

Answer 5

This is theactivity of an enzyme per milligram of total protein

(expressed in μmol min−1mg−1).

Specific activity gives a measurement of enzyme purity in the mixture.

Question 6

What is the rate of a reaction?

Answer 6

Concentration of substrate disappearing (or product produced) per unit time (mol.L−1.s−1).

Question 7

What is the turnover number?

Answer 7

Kcat = Vmax/[enzyme]

Represents the maximum number of reactions catalysed per unit time by each active site

Units of kcat are always reciprocal time i.e. s-1 or min-1

Question 8

What is the M-M equation?

Answer 8

Question 9

What is the LB equation?

Answer 9

Double reciprocal plot:

Question 10

What does the LB plot look like? what are its disadvantages?

Answer 10

Disadvantage: places undue weight on the points obtained at low [S], which are the points where precision is lowest

Question 11

What does the Eadie Hofstee plot equation?

What are its disadvantages?

Answer 11

V is on both axis —> error is multiplied

Question 12

What is the Hanes-Wolf plot?

Answer 12

Question 13

What kind of inhibitor is this?

Answer 13

• Vmax is unchanged
• Km increases

Competitive inhibitor: competes with S to bind to the enzyme. Increasing [S] counteracts the inhibition.

Question 14

What kind of inhibitor is this?

Answer 14

• Vmax is decreased
• Km is unchanged

Non-competitive inhibition: reacts equally well with E or with E-S complex, slows rate of reaction to form E-P. Unlike with a competitive inhibitor, increasing [S] does not relieve inhibition.

Question 15

What kind of inhibitor is this?

Answer 15

• Vmax is reduced
• Km is reduced

Uncompetitive inhibition: inhibitor binds only to E-S, thus reducing Km. However, Vmax is also reduced given that the E-S-I complex only undergoes the reaction to form product slowly.

Question 16

How was the incubation time for the different incubations determined?

Answer 16

Incubation time for the reactions was determined as the time range during which a constant rate of product formation was observed (first order kinetics).

For subsequent reactions, less time was used than that which was identified as the incubation time, because it is best to use as short a time as possible.

Question 17

Why can’t a graph of rate vs. [S] be used to extract Km and Vmax?

Answer 17

It is hyperbolic, Vmax is never fully attained, and Km is a function of Vmax!

Question 18

What were the 5 parts of the practical?

Answer 18

A: determining pH optimum

B: investigating incubation time for reactions

C: investigating the effect of enzyme concentration on reaction rate

D: Investigating the effect of substrate concentration on reaction rate (LB plot)

E: Determining type of inhibition of inhibitors A and B

Question 19

If the requirement is to increase the intracellular concentration of the substrate, what type of inhibitor is needed?

Answer 19

Either a competitive or non-competitive inhibitor will serve, since both will inhibit the utilisation of substrate, so that it accumulates.

Question 20

If the requirement is to decrease the intracellular concentration of the product, what type of inhibitor is required?

Answer 20

Non-competitive. As unused substrate accumulates, so it will compete with a competitive inhibitor, and the final result will be a more or less normal rate of formation of product, but with a larger pool of substrate. Increasing the concentration of substrate does not affect a non-competitive inhibitor.

Question 21

What is Ki?

Answer 21

Ki is the inhibitor constant. It gives an indication of how potent an inhibitor is.

Ki = [I] required to produce half maximum inhibition.

Question 22

What is the Dixon plot?

Answer 22

Plotting 1/v against [I] at each concentration of substrate (the Dixon plot) gives a family of intersecting lines.

Question 23

For a competitive inhibitor, where do lines converge on the Dixon plot?

Answer 23

Above the X-axis. Point where they intersect = -Ki

Question 24

For a non-competitive inhibitor, lines on the Dixon plot converge….

Answer 24

On the X axis. Intercept = -Ki