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Introduction to Biochemical engineering > Enzyme kinetics > Flashcards

Enzyme kinetics Flashcards

1
Q

What are enzymes?

A

Proteins of higher molecular weight that act as biological catalysts - increasing rate of reaction

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2
Q

How are enzymes generally named?

A

By adding -ase to the end of the substrate catalysed (Urease) or the reaction catalysed (alcohol dehydrogenase)

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3
Q

What do enzymes do?

A

Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme-substrate complex.

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4
Q

What are the 6 international classifications of enzymes?

A

Oxidoreductase, Transferases, Hydrolases, Lyases, Isomerases and Ligases

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5
Q

How are enzymes sorted into classifications?

A

Classified according to the reaction they catalyze

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6
Q

What is the simplest model for the interaction between an enzyme and a substrate?

A

Lock and key model

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7
Q

Why is activation energy lower when an enzyme binds to a substrate?

A
  • Enzymes hold the substrates at certain positions and angles to improve the reaction rate (orientation effect)
  • In some enzymes formation of an enzyme-substrate complex causes slight changes in the 3-Dshape of agglomerate which may also promote reactivity
  • In multisubstrate enzyme-catalyzed reactions (enzymes can have more than one active binding site), enzymes can hold substrates so that reactive regions of substrates are close to each other,(proximity effect).
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8
Q

Give 4 examples of some commercial enzymes?

A
  • α-amylase to produce glucose from starch
  • Tyrosine for cheesemaking
  • Proteolytic enzymes for washing powders
  • Glucose isomerase to make fructose from glucose
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9
Q

What is the model of kinetics for simple enzyme-catalysed reactions based on?

A

Data from batch reactors with constant liquid volume in which the initialsubstrate and enzyme concentrations [S0], [E0] are known.

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10
Q

What is the kinetics of simple enzyme-catalysed reactions sometimes called?

A

Michaelis-Menten kinetics or saturation kinetics

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11
Q

How can saturation kinetics be obtained?

A

Saturation kinetics can be obtained from a simple reaction scheme that involves a reversible stepfor enzyme-substrate complex formation, and a dissociation step of the ES complex

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12
Q

What are the two major approaches used in developing a rate expression for the enzyme-catalyzed reactions?

A

Rapid equilibrium approach and quasi-steady-state approach

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13
Q

Both the rapid equilibrium approach and the quasi-steady-state approach have the same initial steps. What are they?

A

v = d[P]/dt = k2[ES]
where v = rate of product formation

d[ES]/dt = k1[E][S] - k-1[ES] - k2[ES]

enzyme in any catalyst is not used so:
[E] = [E0] - [ES]

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14
Q

Use the rapid equilibrium approach to find [ES] in terms of [S]

A
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15
Q

What is K’m?

A

often called the Michaelis-Menten constant, and the prime reminds us that it wasderived by assuming rapid equilibrium in the first step

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16
Q

How does a low K’m suggest?

A

indicates that the enzyme has a high affinity for the substrate

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17
Q

What is Vm?

A

Maximum forward reaction velocity

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18
Q

Draw graph for v against [S]

A
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19
Q

What relation can be made for high [S]?

A

v = Vm

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20
Q

Why was the quasi-steady-state approach introduced?

A

In most cases a closed system (batch reactor) is used in which the initial substrateconcentration greatly exceeds the initial enzyme concentration.

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21
Q

Write the quasi-steady-state approach

A
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22
Q

Describe a brief method of finding Km and Vm from experimental data

A

Typically experimental data are obtained from initial-rate experiments. A batch reactor is charged with a known amount of substrate [S0] and enzyme [E0]. The product (or substrate concentration)is plotted against time. The initial slope of this curve is estimated [i.e. dP/dt (at t=0) = -dS/dt (at t=0)]. This value of v then depends on the values of [E0] and [S0] in the charge to the reactor. Many such experiments can be used to generate many pairs of v and [S] data. These can then be plotted and Km established

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23
Q

Describe the double reciprocal (line-weaver-burk) plot

A
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24
Q

Describe the Eadie-Hofstee plot

A
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25
Q

Describe the Hanes-Woolf plot

A
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26
Q

How can the time course of variation of [S] in a batch enzymatic reactor be determined?

A
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27
Q

What are the limitations of the rapid equilibrium approach and the quasi-steady-state approach?

A

Valid strictly for small enzyme concentration relative to the substrate concentrations

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28
Q

Show that discrepancies occur between the exact solution and the quasi-steady state solution as E0/S0 increases

A
29
Q

Show that there are deviations from Michaelis-Menton kinetics at large values of initial enzyme content

A
30
Q

Name a model for more complex enzyme kinetics regulation

A

Allosteric control

31
Q

What is allostery/cooperative binding?

A

The binding of one substrate to the enzyme facilitates the binding of other substrate molecules as some enzymes have more than one substrate binding site.

32
Q

Give the rate expression for allostery/cooperative binding

A
33
Q

How do you determine the cooperativity coefficient?

A
34
Q

What are enzyme inhibitors?

A

Compounds that bind to enzymes and reduce their activity. These inhibitions may be reversible or irreversible.

35
Q

Give some examples of irreversible enzyme inhibitors and what they do.

A

Form a stable complex with the enzyme and reduce enzyme activity. Lead, Cadmium and Mercury are examples of irreversible enzyme inhibitors

36
Q

What is different about reversible enzyme inhibitors?

A

Dissociate more easily from the enzyme after binding. Usually, special chelating (metal-binding) agents such as citrates

37
Q

What’s the reaction mechanism for a competitive inhibitor?

A
38
Q

find v for a competitive inhibition reaction and therefore show reaction rate decreases as a result of competitive inhibition

A
39
Q

How can competitive inhibition be overcome? Use the Lineweaver-Burk plot

A

High concentrations of substrate

40
Q

What is a non-competitive inhibiting enzyme?

A

Non-competitive inhibitors are not substrate analogues. Inhibitors bind on sites other than the active site and also bind to the free enzyme complex and reduce enzyme affinity to the substrate

41
Q

Find an equation for v for noncompetitive inhibition

A
41
Q

Give the mechanism for a non-competitive inhibitor

A
42
Q

What is the net effect of noncompetitive inhibition? Would high substrate concentration overcome this?

A

The net effect of noncompetitive inhibition is a reduction in Vm. High substrate concentrations would not overcome noncompetitive inhibition. Other reagents need to be added to block binding of the inhibitor to the enzyme

43
Q

Draw double reciprocal (Lineweaver-Burk) for noncompetitive inhibition.

A
44
Q

What is an uncompetitive inhibitor? Give the mechanism.

A

Uncompetitive inhibitors bind to the ES complex only and have no affinity for the enzyme itself.

45
Q

Find an equation for the rate of reaction,v, for uncompetitive substrate inhibtion

A
46
Q

use the equation for the rate of reaction for uncompetitive inhibition to find an equation for max substrate concentration

A
47
Q

What happens to rate of enzyme-catalysed reactions when temperature is increased?

A

Increases, up to a certain limit

48
Q

Why does enzyme activity decrease after a certain temperature

A

enzyme denaturing

49
Q

Give the equation for the rate of enzyme conversion of a substrate, before the peak temperature.

A
50
Q

Give the equation for the rate of enzyme conversion of a substrate, after the peak temperature.

A
51
Q

Draw graph of enzyme activity vs temperature

A
52
Q

What’s the effect of PH on enzyme activity?

A

Variations in the pH of the medium result in changes in the ionic form of the active site and changes in the activity of the enzyme and hence the reaction rate. Changes in pH may also alter the three-dimensional shape of the enzyme. For these reasons, enzymes are only active over a certain pH range. Optimum PH changes enzyme to enzyme.

53
Q

What happens when the substrate is solid?

A

Now we have a solid substrate with a set number of binding sites for the enzyme to bind to. Now the enzyme in solution may equilibrate with bound enzyme and exhibit kinetics which are “opposite” to those found for soluble substances. For example equilibrium adsorption of enzyme [E] onto substrate [S]

54
Q

Write the mechanism for equilibrium adsorption of enzyme [E] onto substrate [S] and find an equation for v

A
55
Q

What is enzyme immobilisation?

A

The restriction of enzyme mobility in a fixed space

56
Q

What are the advantages of enzyme immobilisation?

A
  • Reduce costs of operation compared to free enzyme systems where additional separation and purification steps are needed.
  • Some immobilization methods can increase enzyme activity.
  • A model system to study enzyme action in membrane-bound enzymes that occurs in the cell
57
Q

What are the disadvantages of enzyme immobilisation?

A
  • Many immobilized enzymes exhibit lower activity compared to free enzymes
  • More expensive to prepare than free enzymes
  • Mass transfer limitations due to immobilization methods
58
Q

What are the 2 major categories of enzyme immobilisation?

A

The two major categories of immobilization are entrapment and surface immobilization

59
Q

What are the 2 categories of entrapped immobilised enzymes?

A

Matrix-entrapped and membrane-entrapped

60
Q

What is matrix entrapment?

A

The enzyme solution is mixed with a polymeric fluid that solidifies into various forms, depending on application. The polymeric fluid is semi-permeable. Large molecular weight enzymes cannot diffuse out, but smaller substrate and product molecules can.
Some matrices for entrapment: Agar, Polyacrylamide, Collagen

61
Q

What is membrane entrapment?

A

Enzyme solutions may be confined between thin semi-permeable membranes.
Membrane materials include: Nylon, Polysulfone, Cellulose, Polyacrylate

62
Q

What is the most common membrane configuration?

A

Hollow fibre configuration is a common arrangement for separating enzyme from substrate and product solution.

63
Q

What are the 2 categories of bound enzyme immobilisation?

A

Adsorbed and covalently-bound

64
Q

What is adsorption, related to enzyme immobilisation?

A

Attachment of enzymes to stationary solids by weak physical forces(e.g. van der Waals). A favourable aspect of the weak adsorption forces is that the active site on the enzyme is normally unaffected and nearly full activity is observed. Desorption, however of enzymes is a common problem.
Solid support materials: Alumina, Porous Glass, Diatomaceous Earth (type of rock), Cellulose Materials, Silica, Ceramics, Clay, Activated Carbon, Starch

65
Q

What is covalent bonding, related to enzyme immobilisation?

A

The retention of enzymes on support surfaces by covalent bonding between functional groups on the enzyme and those on the support surface.Functional groups on enzymes: Amino (protein-NH2), Carboxyl (protein-COOH), Hydroxyl (protein-OH), Sulfhydryl (protein-SH)

66
Q

What is the diffusional limitation of enzyme immobilisation?

A

Diffusional limitations are observed to various degrees in all immobilized enzyme systems. This occurs because the substrate must diffuse from the bulk solution up to the surface of the immobilized enzyme prior to the reaction. The rate of diffusion relative to enzyme reaction rate determines whether limitations on intrinsic enzyme kinetics are observed or not

67
Q

What does the Damkohler number indicate and give the equation?

A

whether limitations on intrinsic enzyme kinetics is observed or not in immobilised enzymes

68
Q

List some enzymes used in medicine and give their uses

A

Introduction to Biochemical engineering (18 decks)

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