Enzyme Kinetics Flashcards
What is an Enzyme (E) ?
Protein catalyst
What are Catalysts ?
They speed up reaction without being changed by the reaction
What is a Substrate (S) ?
Reactants in the enzyme catalysed reaction
What is an Active Site ?
Area of the enzyme where substrate binds
How these reactions are controlled and how fast they proceed defines ? give examples
The state of the organism e.g.
- Metabolically active - series of enzyme catalysed reactions are occurring
- Metabolically inactive - the enzyme is slowed down
What can malfunctions in enzyme reactions lead to ? give examples
It can lead to diseases e.g.
- Gout - Xanthine oxidase Catalyses hypoxanthine to H202 and then Xanthine to Uric acid which accumulates in joints
- Porphyria - involves ALA synthase mutations – X-linked disease. Enzyme involved in the synthesis of HEME pathway
- Type 1 Albinism - an eye condition, mainly in males. Tyrosinase enzyme involved in production of melanin (pigment)
- Haemopholia A - FACTOR VIII is a blood clotting factor
What do drugs often do to the enzyme activity ?
It often alters the enzyme activity
What does HIV1 protease target ?
Target for AIDS therapy
What do drugs mimic ?
They mimic a protein chain, but they can not be cleaved by the enzyme and they block activity
Why is HIV1 protease needed ?
HIV1 protease is needed for HIV growth as HIV produces a long polypeptide chain composed of many proteins. The protease then cleaves the protein chain into individual functional proteins
Enzymes are involved in ?
All biological processes
Catalysis offers ?
Control
What is Catalysis ? and what does it need to overcome ? then what does the Enzyme form ?
The acceleration of a chemical reaction by a catalyst. It has to overcome the activation energy which is done by adding an enzyme which lowers it.
Enzyme forms a transition state
Explain an Energetically Favourable Reaction ? and how does the reaction occur ?
The free energy of Y is greater than the free energy of X. Therefore ΔG<0, and the disorder of the universe increases during the reaction X → Y.
And this reaction can occur spontaneously
Explain an Energetically Unfavourable Reaction ? and how does the reaction occur ?
If the reaction X → Y occurred, ΔG would be >0, and the universe would become ordered.
And this reaction can occur only if it is coupled to a second energetically favourable reaction
Define Entropy: 2nd law of thermodynamics ?
Measure of disorder of a system
Explain why Total entropy (S) can never decrease over time for an isolated system ?
The entropy of an isolated system spontaneously evolves toward thermodynamic equilibrium: the entropy should stay the same or increase.
Give the dissociation rate ?
dissociation rate = dissociation rate constant x concentration of AB
Give the association rate ?
association rate = association rate constant x concentration of A x concentration of B
What does binding of enzyme to substrate provide ?
It provides a transition rate.
results in enzyme-product complex (EP)
What happens in an induced fit model ?
It results in a conformational change. Then the enzyme goes back to its original shape
To understand specificity of the active site we need to think about ?
The protein structure
Name some of these ?
- DNA Topoisomerase inhibitors (anticancer)
- Angiotensin converting enzyme inhibitors (blood pressure)
- Xanthine oxidase inhibitors (gout)
- HIV proteinase inhibitors (antiviral)
Enzyme increases ? and reduces ?
It increases the rate of reaction and reduce the activation energy of a reaction making it more rapid
Enzymes do not alter ? and are generally ?
They do not alter the reaction equilibrium and are generally specific for a single chemical reaction
Give the equation of enzyme, substrate and product ?
E + S ↔ ES ↔ EP ↔ E + P
During a reaction many things are changing which provides us with a wide range of things to measure, such as ?
- The decrease in Substrate
- The increase in Product
- (The amount of a side product e.g. H20, H2)
- The amount of time Enzyme is bound to substrate
- The amount of time Enzyme is unbound
Give equation of Catalase reaction and explain why the products are different from substrate ?
2H2O2 (l) ↔ 2H2O(l) + O2(g)
We start with a liquid and get a gas product
What can substrates or products have ?
They can have colour
What does β-lactamase do to penicillin ?
It degrades the penicillin
What is form of penicillin is coloured ?
A modified form of penicillin (Nitrosefin) is coloured
Lots of substrate have and absorb what ?
They have colour so absorb specific wavelength of light
What do many absorb in ?
They absorb in the ultraviolet
So what do we have to use to measure signal ?
A spectrophotometer
When the reaction of Nitrocefin proceeds the colour changes from ?
Yellow to red (abs at 480nm)
What do some enzymes do and give example ?
They alter the size of substrates. For example, DNA restriction endonuclease which cleaves DNA at specific sites along the molecule
To understand enzymes more it’s important to understand how fast it goes with different ?
Different concentrations of substrate available
What do we want to know to characterise an enzyme ?
- The maximum rate of an enzyme catalysed reaction i.e. the fastest that the reaction can go in the presence of the enzyme
- The affinity of the enzyme for substrate i.e. how well the substrate binds to the active site
What quick experiment could we do ? then what do we do once complete ?
Start with high concentration of substrate and MUCH lower concentration of enzyme i.e. excess substrate.
Then measure the rate, now DECREASE the substrate concentration and finally measure it again
To measure how fast the enzyme is processing the substrate which two things could we measure ?
- The decrease in substrate
2. The increase in product
What is Km (Michaelis constant) ?
The concentration of substrate which permits the enzyme to achieve half Vmax
