biology
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Lab6EffectofTemperatureonEnzymeActivity1.pptxIntroduction to Biology BIO 105
Dr. Urbi Ghosh
Lab 4 : Effect of Temperature on Enzyme Activity
Learning Objectives: Enzyme
Importance
How they work
Feedback inhibition
Lock-and-Key model
Induced fit model
Factors that change activity of enzymes:
Temperature
pH
Substrate concentration
Enzyme concentration
A background on enzymes
Define enzymes
Understand concepts such as an enzyme’s active site and enzyme specificity.
Explain the energy of activation.
Describe the structure of enzymes.
Factors that affect enzyme activity:
1. Environmental Conditions (Temperature)
2. Enzyme structure (denaturation)
3. Enzyme Inhibitors
Why are enzymes important?
Enzymes play an important role in Metabolism, Diagnosis, and Therapeutics.
Protein Catalyst
All biochemical reactions are enzyme catalyzed in the living organism.
Level of enzyme in blood are of diagnostic importance e.g. it is a good indicator in disease such as myocardial infarction.
Acts on substrates (reactants).
Catalysts
A catalyst is a molecule or enzyme that increases the rate of a chemical reaction
Lower the activation energy of a reaction.
Not consumed during the reaction.
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How does a catalyst work to lower energy of activation?
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Forms temporary reaction intermediates with reactant molecules to increase rate.
Holds the reactants in the correct orientation for a reaction to go
May weaken or break the bonds with the reactant molecules.
Enzymes
Proteins that catalyze biological reactions
Specific for a particular reaction
Highly specific to the reaction.
The enzyme’s structure determines which reactants it can bind to.
Enzymes can be precisely regulated.
Enzymes
Q. Define enzyme. A. An enzyme is a biological catalyst.
What is so special about an enzyme?
Enzymes are SPECIFIC.
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Enzyme Specificity
Enzymes have varying degrees of specificity for substrates
The structure of enzymes relates to the substrate it binds to.
Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of bond
Enzyme Substrate Complex
“Lock-and-Key”
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Enzymes
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This Photo by Unknown Author is licensed under CC BY-SA
Almost all reactions in the body are controlled by enzymes
All enzymes are proteins (built with amino acids)
Enzymes are special protein which control cellular reactions.
Enzymes can be used over and over again (reusable).
Enzymes are specific: many different enzymes are required to break down food (carbohydrate, fats and proteins)
Enzymes are special protein which control cellular reactions.
As body conditions change, the reactivity and structure of enzyme changes.
The enzyme’s activity is determined by feedback mechanisms.
The feedback mechanisms allows for precise regulation in changing body conditions.
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Enzyme Inhibitors
Competive - mimic substrate, may block active site, but may dislodge it.
Catalase enzyme
Hydrogen peroxide is a harmful byproduct of many normal metabolic processes; to prevent damage to cells and tissues, it must be quickly converted into other, less dangerous substances.
To this end, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less-reactive gaseous oxygen and water molecules.
Catalase
Catalase is an enzyme found in almost every living tissue.
The large majority of known organisms use catalase in every organ, with particularly high concentrations occurring in the liver in mammals
Catalase catalyzes the following reaction:
It is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS).
Catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of hydrogen peroxide molecules to water and oxygen each second.
Enzyme structure and specificity
Active site: The area on the enzyme where the substrate or substrates attach to is called the active site.
Enzymes are usually very large proteins and the active site is just a small region of the enzyme molecule.
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Enzymes: Active sites
Enzyme molecules contain a special pocket or cleft called the active sites.
In enzymatic reactions, the substance at the beginning of the process, on which an enzyme begins it’s action is called substrate.
Lock-and-Key Model
In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the active site
This is an older model, however, and does not work for all enzymes
How does enzymes lower energy of activation?
Enzymes are proteins that increase the rate of reaction HOW? by lowering the energy of activation
They catalyze nearly all the chemical reactions taking place in the cells of the body.
Not altered or consumed during reaction.
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Enzymes Lower a Reaction’s Activation Energy
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Energy and Enzymes
6/18/2020
G. Podgorski, Biol. 1010
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Activation energy is the push needed to start a reaction
Enzyme Catalyzed Reactions
When a substrate (S) fits properly in an active site, an enzyme-substrate (ES) complex is formed:
E + S ES
Within the active site of the ES complex, the reaction occurs to convert substrate to product (P):
ES E + P
The products are then released, allowing another substrate molecule to bind the enzyme
- this cycle can be repeated millions (or even more) times per minute
The overall reaction for the conversion of substrate to product can be written as follows:
E + S ES E + P
Enzyme-substrate complex
Step 1:
Enzyme and substrate combine to form complex
E + S ES
Enzyme Substrate Complex
+
+
Enzyme-product complex
Step 2:
An enzyme-product complex is formed.
ES EP
ES
EP
transition state
Within the active site of the ES complex, the reaction occurs to convert substrate to product (P):
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Product
The enzyme and product separate
EP E + P
The product
is made
Enzyme is ready
for
another
substrate.
EP
The products are then released, allowing another substrate molecule to bind the enzyme
- this cycle can be repeated millions (or even more) times per minute
The overall reaction for the conversion of substrate to product can be written as follows:
E + S ES E + P
The enzyme Product
The enzyme and product separate
EP E + P
EP
Enzyme is ready
for
another
substrate.
The product
is made
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Structure of enzymes
Enzymes
Complex or holoenzymes (protein part and nonprotein part – cofactor)
Simple (only protein)
Apoenzyme (protein part)
Cofactor
Prosthetic groups
usually small inorganic molecule or atom;
usually tightly bound to apoenzyme
Coenzyme
-large organic molecule
-loosely bound to apoenzyme
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Important Terms to Understand Biochemical Nature And Activity of Enzymes
Cofactor:
A cofactor is a non-protein chemical compound that is bound (either tightly or loosely) to an enzyme and is required for catalysis.
Types of Cofactors:
Coenzymes.
Prosthetic groups.
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What Factors Affect Enzyme Activity?
Temperature
pH
Substrate concentration
Enzyme concentration
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Environmental Conditions
1. Extreme Temperature are the most dangerous
- high temps may denature (unfold) the enzyme.
2. pH (most like 6 - 8 pH near neutral)
3. substrate concentration .
Temperature and enzymes
Optimum temperature The temp at which enzymatic reaction occur fastest.
Q. Define optimum temperature. A. Optimum temperature is the temperature at which the enzyme has it maximum activity (highest rate of reaction)
The temp at which enzymatic reaction occur fastest is called Optimum temperature
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Temperature effect on enzyme
Temperature affects the reaction rate of enzymes
1. At low temperatures, enzymes have low activity. As the temperature rises the rate of reaction increases, usually 2-fold for every 10 degree Celsius rise.
2. The activity peaks at a specific temperature unique to the enzyme. This is known as the optimum temperature - the temperature at which an enzyme is maximally active.
3. Beyond the optimum temperature the activity of the enzyme decreases.
At extreme temperatures, the enzymes are denatured and activity ceases.
Temperature and enzymatic activity
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Q. Account for the increase in slope/ the rate of reaction before the optimal temperature is attained.
A. Temperature is a measure of the kinetic energy of the molecules in a system. As the temperature increases, the kinetic energy and thus the number of random collisions of enzyme with substrate increases per unit time.
With the increase in frequency in the number of collisions, the probability of the enzyme and substrate colliding in the correct orientation (ie. the substrate fits into the active site) also increases.
Hence rate of reaction increases with temperature increases, up until the optimum temperature is attained.
Q. Account for the decline in the slope after the optimal temperature is attained.
A. A further increase in the temperature beyond the optimal temperature leads to disruption of the weak bonds of the enzyme - enzymes are proteins. The unfolding of the protein due to the disruption of bonds is called denaturing. During denaturation the precise configuration of the active site is lost. As the active sites are lost, the catalytic activity and hence rate of reaction decrease.
Catalase
Catalase is an enzyme found in almost every living tissue.
The large majority of known organisms use catalase in every organ, with particularly high concentrations occurring in the liver in mammals
Catalase catalyzes the following reaction:
It is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS).
Catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of hydrogen peroxide molecules to water and oxygen each second.
Catalase
This Photo by Unknown Author is licensed under CC BY-NC-ND
Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long
In the following experiment the activity of catalase is measured and graphed over a range of temperatures.
The substrate for catalase is hydrogen peroxide and the products of its decomposition are water and oxygen.
Hydrogen peroxide is a reactive oxygen species and by-product of several biochemical reactions. Since hydrogen peroxide becomes toxic if it accumulates, catalase protects cells from oxidative damage. The rate of reaction is monitored by the rate of appearance of the product oxygen.
What is the effect of temperature on catalase?
When temperature is above or below _______C , the catalase produced less oxygen.
The catalase made the chemical reaction produce less oxygen.
Thus the optimal temperature for catalase is ________
2. Did the boiled catalase behave differently that non-boiled catalase? Explain
Yes
The boiled catalase produced less oxygen than the non boiled catalase at the room temperature
Why ?
Because the catalase enzyme was denatured when boiled.
Denaturation of enzyme
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3. What conditions needed to remain constant for each trial?
The conditions that needed to remain constant are the amount of hydrogen peroxide, catalase and water temperature.
Taking the same amount of hydrogen peroxide each time
Taking the same amount of catalase every time.
Substrate Concentration and Reaction Rate
The rate of reaction increases as substrate concentration increases (at constant enzyme concentration)
Maximum activity occurs when the enzyme is saturated (when all enzymes are binding substrate)
Enzymes and pH
This Photo by Unknown Author is licensed under CC BY-SA
Lock-and-Key Model
In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the active site
This explains enzyme specificity
This explains the loss of activity when enzymes denature
Induced Fit Model
In the induced-fit model of enzyme action:
- the active site is flexible, not rigid
- the shapes of the enzyme, active site, and substrate adjust to maximumize the fit, which improves catalysis
- there is a greater range of substrate specificity
This model is more consistent with a wider range of enzymes
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2. Cofactors and Coenzymes
Inorganic substances (zinc, iron) and vitamins (respectively) are sometimes need for proper enzymatic activity.
Example:
Iron must be present in the quaternary structure - hemoglobin in order for it to pick up oxygen.
Enzymes
Proteins that catalyze biological reactions
Specific for a particular reaction
Highly specific to the reaction.
The enzyme’s structure determines which reactants it can bind to.
Enzymes can be precisely regulated.
