Enzyme kinetics

Question 1

What is enzyme activity?

Answer 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.

(In scientific literature you will sometimes see the SI unit ‘katal’ used. 1 katal = 1 mol.s-1, but this is an excessively large unit and so is not so common as U).

Question 2

What is specific activity?

Answer 2

This is the activity of an enzyme per milligram of total protein (expressed in μmol.min-1.mg-1).

Specific activity gives a measurement of the activity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of total protein.

Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein.

Question 3

What is the rate of reaction?

Answer 3

The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol.L−1.s−1).

Question 4

How is the % purity calculated?

Answer 4

The % purity is

100% × (specific activity of enzyme sample / specific activity of pure enzyme)

The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated.

Question 5

What is the M-M equation?

Answer 5

V = (Vmax*[S])/ (Km + [S])

Question 6

What does the M-M graph look like?

Answer 6

Question 7

In M-M kinetics, what are Vmax and Km?

Answer 7

Vmax: limitng rate. Rate of reaction when enzyme is saturated.

Km: [S] at which enzyme is half-saturated, and V is 1/2*Vmax

Question 8

What is Kcat?

Answer 8

Catalytic constant of an enzyme, also referred to as the turnover number.

Represents the maximum number of reactions catalysed per unit time by each active site. To calculate it you need to know the Vmax and the enzyme concentration in the same concentration units. In this case kcat = Vmax divided by the enzyme conc. e.g. if the rate is 500 μM.s-1 at an enzyme concentration 1 μM, kcat will be 500 s-1.

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

Question 9

If an enzyme assay was carried out spectrophotometrically, how is Vmax in deltaA/min converted into microM/min?

Answer 9

Question 10

What are the units of the molar extinction coefficient?

Answer 10

Lmol-1cm-1.

To work it out: remember that absorbance is unitless.

Question 11

What is the pKa?

Answer 11

Defined as:

pKa = -log(Ka)

It is the pH at which an ionisable species (e.g carboxylic acid, or an ionisable group on an aa backbone or side chain) is half ionised

Question 12

What is the LB equation?

Answer 12

Question 13

What does the LB plot look like?

Answer 13

Question 14

Which side of a reaction will be favoured if

a) Ke >> 1
b) Ke << 1

Answer 14

a) products
b) reagents

Question 15

What is the H-H equation?

Answer 15

Question 16

What is isoelectric focusing?

Answer 16

Protein separation technique which exploits differences in pI values.

Proteins are immobilised at different points in the pH gradient according to their individual pI values.

Question 17

What is dyalisis used for?

Answer 17

Dialysis is mainly used to remove small molecules such as ions from the protein mixture following procedures such as salting out.

Question 18

What is differential centrifugation used for?

Answer 18

Differential centrifugation provides a method to isolate protein mixtures from cellular components and debris in cell extracts, but it would not be useful to separate the two proteins as they have very similar molecular weights (240 kDa for catalase and 160 kDa for glucose oxidase) and thus would fail to separate adequately by centrifugation.

Question 19

Why is a long, thin column used for gel filtration ?

Answer 19

Gel filtration columns ought to be long and narrow in order to slow down the flow rates and increase resolution. This is due to the fact that slow rates, achieved by suitable column dimensions, allow efficient partitioning into pores and thus increase the resolution.

Question 20

What is the exclusion limit of a gel filtration column?

Answer 20

Max Mr (in kDa) above which proteins are excluded from penetrating the gel matrix.

Question 21

If the pKa is determined spectrophotometrically, what is the best way to manipulate the data to estimate it?

Answer 21

It can be done from a titration curve (absorbance vs. pH), but it’s best to do it with a linearised plot:

pH vs. log ([A^-]/([T] - [A^-]))

Let [T] = the TOTAL concentration of the weak acid i.e. [T] = [A⁻] + [HA] [HA] = ([T] - [A⁻])

Hence, pH = pKa + log ([A⁻]/([T] - [A⁻]))

pKa will be the inercept on y axis