Enzyme kinetics Flashcards
What are enzymes?
Proteins of higher molecular weight that act as biological catalysts - increasing rate of reaction
How are enzymes generally named?
By adding -ase to the end of the substrate catalysed (Urease) or the reaction catalysed (alcohol dehydrogenase)
What do enzymes do?
Enzymes lower the activation energy of the reaction catalyzed by binding the substrate and forming an enzyme-substrate complex.
What are the 6 international classifications of enzymes?
Oxidoreductase, Transferases, Hydrolases, Lyases, Isomerases and Ligases
How are enzymes sorted into classifications?
Classified according to the reaction they catalyze
What is the simplest model for the interaction between an enzyme and a substrate?
Lock and key model
Why is activation energy lower when an enzyme binds to a substrate?
- 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).
Give 4 examples of some commercial enzymes?
- α-amylase to produce glucose from starch
- Tyrosine for cheesemaking
- Proteolytic enzymes for washing powders
- Glucose isomerase to make fructose from glucose
What is the model of kinetics for simple enzyme-catalysed reactions based on?
Data from batch reactors with constant liquid volume in which the initialsubstrate and enzyme concentrations [S0], [E0] are known.
What is the kinetics of simple enzyme-catalysed reactions sometimes called?
Michaelis-Menten kinetics or saturation kinetics
How can saturation kinetics be obtained?
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
What are the two major approaches used in developing a rate expression for the enzyme-catalyzed reactions?
Rapid equilibrium approach and quasi-steady-state approach
Both the rapid equilibrium approach and the quasi-steady-state approach have the same initial steps. What are they?
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]
Use the rapid equilibrium approach to find [ES] in terms of [S]
What is K’m?
often called the Michaelis-Menten constant, and the prime reminds us that it wasderived by assuming rapid equilibrium in the first step
How does a low K’m suggest?
indicates that the enzyme has a high affinity for the substrate
What is Vm?
Maximum forward reaction velocity
Draw graph for v against [S]
What relation can be made for high [S]?
v = Vm
Why was the quasi-steady-state approach introduced?
In most cases a closed system (batch reactor) is used in which the initial substrateconcentration greatly exceeds the initial enzyme concentration.
Write the quasi-steady-state approach
Describe a brief method of finding Km and Vm from experimental data
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
Describe the double reciprocal (line-weaver-burk) plot
Describe the Eadie-Hofstee plot
Describe the Hanes-Woolf plot
How can the time course of variation of [S] in a batch enzymatic reactor be determined?
What are the limitations of the rapid equilibrium approach and the quasi-steady-state approach?
Valid strictly for small enzyme concentration relative to the substrate concentrations
Show that discrepancies occur between the exact solution and the quasi-steady state solution as E0/S0 increases
Show that there are deviations from Michaelis-Menton kinetics at large values of initial enzyme content
Name a model for more complex enzyme kinetics regulation
Allosteric control
What is allostery/cooperative binding?
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.
Give the rate expression for allostery/cooperative binding
How do you determine the cooperativity coefficient?
What are enzyme inhibitors?
Compounds that bind to enzymes and reduce their activity. These inhibitions may be reversible or irreversible.
Give some examples of irreversible enzyme inhibitors and what they do.
Form a stable complex with the enzyme and reduce enzyme activity. Lead, Cadmium and Mercury are examples of irreversible enzyme inhibitors
What is different about reversible enzyme inhibitors?
Dissociate more easily from the enzyme after binding. Usually, special chelating (metal-binding) agents such as citrates
What’s the reaction mechanism for a competitive inhibitor?
find v for a competitive inhibition reaction and therefore show reaction rate decreases as a result of competitive inhibition
How can competitive inhibition be overcome? Use the Lineweaver-Burk plot
High concentrations of substrate
What is a non-competitive inhibiting enzyme?
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
Find an equation for v for noncompetitive inhibition
Give the mechanism for a non-competitive inhibitor
What is the net effect of noncompetitive inhibition? Would high substrate concentration overcome this?
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
Draw double reciprocal (Lineweaver-Burk) for noncompetitive inhibition.
What is an uncompetitive inhibitor? Give the mechanism.
Uncompetitive inhibitors bind to the ES complex only and have no affinity for the enzyme itself.
Find an equation for the rate of reaction,v, for uncompetitive substrate inhibtion
use the equation for the rate of reaction for uncompetitive inhibition to find an equation for max substrate concentration
What happens to rate of enzyme-catalysed reactions when temperature is increased?
Increases, up to a certain limit
Why does enzyme activity decrease after a certain temperature
enzyme denaturing
Give the equation for the rate of enzyme conversion of a substrate, before the peak temperature.
Give the equation for the rate of enzyme conversion of a substrate, after the peak temperature.
Draw graph of enzyme activity vs temperature
What’s the effect of PH on enzyme activity?
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.
What happens when the substrate is solid?
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]
Write the mechanism for equilibrium adsorption of enzyme [E] onto substrate [S] and find an equation for v
What is enzyme immobilisation?
The restriction of enzyme mobility in a fixed space
What are the advantages of enzyme immobilisation?
- 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
What are the disadvantages of enzyme immobilisation?
- Many immobilized enzymes exhibit lower activity compared to free enzymes
- More expensive to prepare than free enzymes
- Mass transfer limitations due to immobilization methods
What are the 2 major categories of enzyme immobilisation?
The two major categories of immobilization are entrapment and surface immobilization
What are the 2 categories of entrapped immobilised enzymes?
Matrix-entrapped and membrane-entrapped
What is matrix entrapment?
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
What is membrane entrapment?
Enzyme solutions may be confined between thin semi-permeable membranes.
Membrane materials include: Nylon, Polysulfone, Cellulose, Polyacrylate
What is the most common membrane configuration?
Hollow fibre configuration is a common arrangement for separating enzyme from substrate and product solution.
What are the 2 categories of bound enzyme immobilisation?
Adsorbed and covalently-bound
What is adsorption, related to enzyme immobilisation?
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
What is covalent bonding, related to enzyme immobilisation?
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)
What is the diffusional limitation of enzyme immobilisation?
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
What does the Damkohler number indicate and give the equation?
whether limitations on intrinsic enzyme kinetics is observed or not in immobilised enzymes
List some enzymes used in medicine and give their uses
