Experiment D: enzyme assay Flashcards
regarding pH, when does an enzyme show maximum activity?
at the optimum pH
why does pH affect enzymatic activity?
- substrate binding involves interaction with amino acid side chains at the active site
- the pH may affect the ionisation of the substrate and also of the side chains at the active site, thus affecting binding
what is the effect of extreme values of pH?
extreme values of pH may disrupt the tertiary structure of the enzyme to distort the active site and even denature the protein
what is the effect of having mainly uncharged reactive groups at the active site?
the enzyme has activity over a relatively broad range of pH values
what is the plasma or intracellular pH?
pH 7.35-7.45
how do enzymes with very different pH optima to the intracellular average maintain their activity?
by sub-cellular compartmentation
how should the activity of an enzyme be determined with respect to pH?
the activity of an enzyme should be determined near or at the optimum pH to produce meaningful results
what is the relationship between incubation time and amount of product formed?
the longer an enzyme is incubated with its substrate, the greater the amount of product formed
true or false: the rate of product formation is a simple function of incubation time
false; all proteins suffer denaturation, and hence loss of catalytic activity, over time
are enzyme-catalysed reactions reversible or irreversible?
reversible
how does the reversibility of enzyme-catalysed reactions affect the formation of product?
- initially, there is little or no product present, so the reaction proceeds only in the forward direction
- as the reaction continues, there is a significant accumulation of product and therefore a significant rate of back reaction
- therefore, the rate of product formation decreases as the incubation proceeds
what is the effect of having too long an incubation time?
the measured activity of the enzyme is falsely low
how is the rate of product formation measured regarding incubation time?
- measurements made at short time intervals (eg. 10 seconds) for the first minute or so
- graph plotted of amount of product formed against incubation time
- initial rate determined by drawing a tangent to the steepest part of the curve
what is the problem with short incubation times?
- considerable error of timing
- only a small amount of product is formed so analytical errors are magnified
what incubation time should be used for enzyme-catalysed reactions?
- long enough to allow a moderate amount of product to be formed
- long enough so that an error in timing is insignificant
- not so long that there is a detectable levelling-off of the curve
what must you ensure when determining the rate of reaction?
that the enzyme activity has been constant throughout the incubation
what effect does concentration have on some monomer enzymes?
some enzymes form dimers or other aggregates at high concentrations, affecting the catalytic activity
what effect does concentration have on some enzymes that have a natural form of dimers or other aggregates?
these enzymes may disaggregate when the preparation is diluted, affecting the catalytic activity
how can the ready denaturation in dilute solution of some enzymes be countered?
by adding a relatively large amount of inert protein, such as albumin, to act as a chaperone to the purified complex
what would be the effect of product formation other than that as a result of enzyme activity?
- graph of product formed against enzyme concentration
- constant gradient
- y-intercept above zero, since some product is present even when there is no enzyme present
what amount of enzyme should be used?
- enzyme preparation is generally the most valuable constituent of the incubation
- lowest amount of enzyme that can be pipetted with acceptable precision
- and that leads to the formation of a readily detectable amount of product
how is the activity of an unpurified enzyme expressed?
mol of product formed / unit time / volume of preparation
mol of product formed / unit time / mass of original tissue (g)
how is the activity of a partially purified enzyme expressed?
specific activity;
mol of product formed / unit time / mass of protein (mg)
how is the activity of a purified enzyme expressed?
mol of product formed / time (s) / mol of enzyme
if the molecular mass is known
what is the effect of temperature on an enzyme-catalysed reaction?
- as the temperature increases, the rate increases
- since more molecules have an energy at/above the activation energy
- sharp increase in the formation of product 5-50C
BUT - because enzymes are proteins, they are denatured by heat
- at higher temperatures, there is a rapid loss of activity as the protein suffers irreversible denaturation
why is it not useful to determine the ‘optimum’ temperature for an enzyme-catalysed reaction?
the longer the incubation time, the lower the temperature at which there is maximum formation of product, because of the greater effect of denaturation of the enzyme
what is the conventional temperature used for enzyme-catalysed reactions?
30C; this is a compromise between
- mammalian/clinical biochemists - 37C
- microbial biochemists - 20C
what is the Arrhenius equation?
k = AE^-Ea/RT
when is k equal to Vmax?
for temperatures with significant denaturation, if the enzyme is saturated with substrate
what is the relationship between rate of reaction and the concentration of substrate?
there is a hyperbolic relationship between the two variables
what is the limiting factor at low substrate concentrations?
the concentration of available substrate
what is the limiting factor at increasing substrate concentrations?
the enzyme activity
what is the rate of reaction when the enzyme is saturated with substrate?
Vmax
what is Km?
- the concentration of substrate that allows the enzyme to achieve half Vmax
- the concentration of substrate at which the enzyme is at half-saturation
what does it mean if an enzyme has a relatively high Km?
- low affinity for its substrate
- requires a greater concentration of substrate to achieve Vmax
- not normally saturated with substrate
- activity will vary as the substrate concentration varies
what does it mean if an enzyme has a relatively low Km?
- high affinity for its substrate
- acts at an approximately constant rate
under which conditions can the amount of enzyme in a sample be determined?
- the limiting factor must be the enzyme activity
- and not the amount of available substrate
- concentration of substrate must be high enough to ensure that the enzyme is acting at Vmax
what is the amount of enzyme generally used to determine the activity for an enzyme-catalysed reaction?
about 10-20-fold higher than the Km
under which conditions can the concentration of substrate be determined for an enzyme-catalysed reaction?
- the substrate must be the limiting factor
- the concentration of substrate must be below Km
- so that the rate of product formation increases steeply with increasing substrate concentration
outline the features of a Lineweaver-Burk plot
1/V = 1/Vmax + Km/Vmax (1/[S]) plotting 1/V against 1/[S] , y-intercept = 1/Vmax x-intercept = - 1/Km gradient = Km/Vmax
what is a disadvantage of the L-B plot?
- places undue weight on points obtained at low concentrations of substrate,
- at which precision of determining the rate of reaction is lowest
- because of the small amount of product that has been formed
outline the features of a Eadie-Hofstee plot
V = Vmax - Km (V/[S]) plotting V against V/[S] , y-intercept = Vmax x-intercept = Vmax/Km gradient = - Km
what are the dis/advantages of the Eadie-Hofstee plot?
advantage: overcomes the problem of uneven spacing of points and undue weight given to low values of [S]
disadvantage: V (the dependent variable) is used on both axes; errors in measuring rate of reaction are multiplied, resulting in lower precision of Km and Vmax estimates
outline the features of a Hanes plot
[S]/V = Km/Vmax + [S] (1/Vmax) plotting [S]/V against [S] , y-intercept = Km/Vmax x-intercept = - Km gradient = 1/Vmax
what are the dis/advantages of a Hanes plot?
advantage: overcomes the problem of uneven spacing of points and undue weight given to low values of [S]
disadvantage: [S] (the dependent variable) is used on both axes; errors in measuring rate of reaction are multiplied, resulting in lower precision of Km and Vmax estimates
explain competitive inhibition and its effect on an enzyme-catalysed reaction
- inhibitor competes with the substrate for the active site
- Vmax is unchanged
- Km is increased
- gradient is increased
explain why Vmax is unchanged but Km appears increased on addition of a competitive inhibitor
Vmax - if the enzyme concentration is sufficiently high, the enzyme will still reach full saturation
Km - more substrate is required to reach half saturation; though the affinity of the enzyme is not affected
explain non-competitive inhibition and its effect on an enzyme-catalysed reaction
- inhibitor binds to both the enzyme and to the enzyme-substrate complex
- Vmax is decreased
- Km is unchanged
- gradient is increased
explain why Km is unchanged but Vmax is decreased on addition of a non-competitive inhibitor
Km - the enzyme has the same affinity for the enzyme
Vmax - but the reaction cannot reach Vmax
explain uncompetitive inhibition and its effect on an enzyme-catalysed reaction
- inhibitor binds only to the enzyme-substrate complex
- Vmax is decreased
- Km is decreased
- gradient remains constant
explain why both Vmax and Km are decreased on addition of an uncompetitive inhibitor
Vmax - the reaction cannot reach Vmax
Km - the affinity of the enzyme is increased
explain why the gradient of a L-B plot remains the same on addition of an uncompetitive inhibitor
Vmax and Km decrease to the same extent
what is Ki?
the inhibition constant;
- indication of how potent an inhibitor is
- the concentration of substrate required to produce half-maximum inhibition
what is a Dixon plot?
- plot 1/V against concentration of inhibitor at each substrate concentration
- gives a family of intersecting lines
what is the point of convergence of a Dixon plot for a competitive inhibitor?
- convergence above the x-axis
- at an x-value of -Ki
what is the point of convergence of a Dixon plot for a non-competitive inhibitor?
- convergence on the x-axis
- intersect is -Ki
what incubation time was used in the experiment?
10 minutes
what amount of enzyme was used in the experiment?
100 μL
what is the effect of an enzyme on the energy of the reaction?
- lowers the energy of the transition state
- raises the energy of the ground state
