Practical D Flashcards

1
Q

What 5 properties do enzymes differ in?

A
  1. optimum pH
  2. Vmax
  3. Km (affinity for substrate)
  4. Temperature dependence and thermal denaturing
  5. Sensitivity to inhibitor
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2
Q

How were each of the 5 prpoerties changed in the practical?

A
  1. pH: 1-14
  2. Time: 0-60mins
  3. Enzyme concentration: 0-250 micromolar
  4. Incubation tempurature: 0-100 degrees
  5. Substrate concentration 0-250 millimolar
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3
Q

What is the optimum pH of an enzyme?

A

The pH at which the enzyme is at its maximum acticity

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4
Q

How does pH affect the formation of the enzyme-substrate complex?

A

BInding is dependent on the complementary of side chains of amino acids on the substrate and active site of enzyme
Changes in pH alter the ionisation of these side chains affecting binding
pH can also change the tertiary structure of the active site

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5
Q

What is the relative range of pH activity for chargd and uncharged side chains?

A

Uncharged chains have a broader pH range compared to charged chains i.e they are more sensitive the changes to in pH

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6
Q

How are enzymes, whose optimum pH differs from the intracellur pH, function?

A

Subcellular compartmentation allows the enzymes to function at its own optimum pH

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7
Q

At what pH should enzyme activity be determined?

A

Enzyme activity must be determined at the optimum pH

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8
Q

Descibe the relationship between incubation time and product formed?

A

The longer the incubation time, the more product form.
The relationship is not linear but hyperbolic.
Initial rate is far grater

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9
Q

What happens to all enzymes of time?

A

All enzymes denature over time

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10
Q

Why must enzyme activity be measured using the intial rate?

A

Even if the [Substrate] saturates the enzyme, most of it will be consumed at the start of the reaction so less substrate will be available decreasing teh rate of product formed.
Also as the reaction progresses, the reverse reaction increases

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11
Q

How do you determine the intial rate from the graph?

A

Plot the rate of product formed over the first few seconds.

Draw a tangent to the steepest part of the curve

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12
Q

What is the error associated with a short incubation time?

A

The concentration of the product at the start is very low so it difficult to measure the amount of product precisely resulting in analytical errors

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13
Q

What is the best incubation time?

A

A duration which allows for accurate measuring of the product ( insignificant analytical errors).
Duration is not too long that the curve begins to slope

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14
Q

What happens to enzymes at high concentrations?

A

Enzymes may aggregate or form dimers affecting the activity

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15
Q

What can happen to an enzyme at very low concentrations ?

A

Enzymes that are natural dimers/oligomers may dissociate changing enzyme activity

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16
Q

What can be the problem with low concentrations of an enzyme. What is done to overcome this problem?

A

If an enxyme is unstable at low concs, it may readily denature lowering enzyme activity. Inert enzyme is added to act as chaperone for purified enzyme

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17
Q

Why might a [enzyme],[substrate] graph not pass through the origin?

A

The product is being produce from an alternative pathway which does not involve the investigated enzyme

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18
Q

What amount of enzyme is used and why?

A

The smallest pippetable amount
Purified enzyme is most valuable as it may a very long time to prepare or may have been extracted from a very small sample of tissue

19
Q

What units are used for unpurified/ partially purified enzyme?

A

mol (product formed)/time/volume (of preparation)

mol (product formed)/time/mg of protein

20
Q

What units are used for a purified protein?

A

Given as the catalytic constant, kcat

mol (product formed)/sec/mol of enzyme

21
Q

What are the effects of temperature?

A

Temperature changes the proprtion of particles with energy greater than the activation energy.
Temperature affects the tertiary structure of the protein with high temperatures resulting in the denaturing of the proteins

22
Q

What temperature is used for most enzyme experiments?

A

30 degrees

23
Q

What is Arrhenius’ Equation?

A

k=Ae^-(Ea/RT) or

lnk=lnA-ln(Ea/RT)

24
Q

What is the form of the Arrhenius equation if Temp is very high?

A

k=Vmax

25
Q

Draw a typical enzyme substrate graph

A

Draw a hyperbolic graph with high initial rate with the curve sloping at high [substrates]

26
Q

What is Vmax, Units?

A

The rate of an ezyme when saturated.

mol/s

27
Q

What is Michaellis’ constant? What does it show?

A

Km is the concentration of substrate needed for an enzyme to reach half Vmax.
Shows the affinity of the enzyme for the substrate.
The lower th Km value, the greater affinity

28
Q

Is Vmax a constant? Why?

A

Vmax is not a constant as its value depends on the concentration of enzyme available.

29
Q

What is needed in order to determine the amount of enzyme present?

A

The rate of product formed must be constant so the substrate concentrations must be high enough that rate does not change.
A [substrate] 10 to 20 times the Km is normally used

30
Q

What is needed in order to determine the amount of substrate present?

A

Substrate concentraation must be the limiting factor so concs of subtrate far lower than Km is used

31
Q

What is the equation for V?

A

V= Vmax x [S] / Km + [S]

32
Q

What is the equation for Kcat?

A

Kcat= Vmax/[E]

33
Q

What do the x, y, gradient show on the linweaver burke plot?

A

1/v=1/Vmax + Km/Vmax x 1/[S].
X intercept = -1/Km
y intercept= 1/Vmax
Gradient = Km/Vmax

34
Q

What’s the disadvantage of using LW-B plot?

A

Unfair weight on points initial rate where measurements of product formed are less precise

35
Q

What does the Eadle-Hofsted plot show?

A

v=Vmax-Km(v/[S])
y= Vmax
x= Vmax/km
gradient= -km

36
Q

What are the disadvantages of using E-H plot?

A

Dependent variable is on both axis, thus effect of error of measuring rate of product formed is multiplied.
Lower precision of Km and Vmax

37
Q

What is the Hanes plot?

A
[S]/v= Km/Vmax + [S]/Vmax. PLot [S]/v against [S].
y= Km/Vmax
x= -Km
gradient= 1/Vmax
38
Q

What is the disadvantage og Hanes plot?

A

Independent variable is on both axis, thus effect of error of measuring [S] is multiplied.
Lower precision of Km and Vmax

39
Q

How do competitive inhibitors work? Are they reversible? Why?

A

Comp inh bind to active site. Occupying site

Reversible, increasing [Substrate] lowers effect of inh

40
Q

What is the effect of competitive inhibitor on LW-B plot?

What does it show?

A

Y intercept is unaltered: same Vmax. This is because V will increase to Vmax eventually after more substrate is added.
X intercept is greater, shifts to the right. Km is greate. This represents that a greater concentration is needed to reach half Vmax.
Gradient is increased as Km is larger while Vmax remains the same

41
Q

What is the effect of non-competitive inhibitor on LW-B plot?
What does it show?

A

Y intercept is higher. Vmax is lower. Shows that inhibtior is irreversible. Will ever reach original Vmax
X intercept remains the same.
Gradient is decreased as Vmax is increased

42
Q

How do non-competitive inhibitors work? Are they reversible? Why?

A

Non-comp is irreversible. Binding of inh to surface of enzyme permenantly altering the active site

43
Q

What is the inhibition constant

A

Ki: conc of inh that will produce half of the maximum inhibition

44
Q

What is the Dixon plot?

What is the effect of comp/non-comp on the plot

A

1/v against concentration of inhibitor
comp inh: lines converge above x axis at value of -ki
non-comp: ines converge at x axis. intercept is -ki