Enzyme kinetics Flashcards
What is enzyme activity?
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).
What is specific activity?
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.
What is the rate of reaction?
The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol.L−1.s−1).
How is the % purity calculated?
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.
What is the M-M equation?
V = (Vmax*[S])/ (Km + [S])
What does the M-M graph look like?
In M-M kinetics, what are Vmax and Km?
Vmax: limitng rate. Rate of reaction when enzyme is saturated.
Km: [S] at which enzyme is half-saturated, and V is 1/2*Vmax
What is Kcat?
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
If an enzyme assay was carried out spectrophotometrically, how is Vmax in deltaA/min converted into microM/min?
What are the units of the molar extinction coefficient?
To work it out: remember that absorbance is unitless.
What is the pKa?
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
What is the LB equation?
What does the LB plot look like?
Which side of a reaction will be favoured if
a) Ke >> 1
b) Ke << 1
What is the H-H equation?