MB+B Enzymes Flashcards
What are the 6 types of enzyme
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
What is Keq
The equalibrium constant = [B]/[A]
Why is the relationship between substrate concentration and rate non-linear
The reaction becomes saturated and the enzyme becomes the limiting factor and reaction slows as it approaches maximum value
What is Vmax
The maximum rate a reaction can achieve
What is Km
Michaelis constant = Vmax/2
When is the michaelis menten equation used
To get an accurate measurement of Km
What is the michaelis menten equation
V= Vmax x [S] / Km + [S]
What assumptions does the michaelis menten equation rely on
The step from the formation of the enzyme substrate complex to product is irresversable
Substrate is in abundance
ESC is at a steady state (unchangeable over time)
What is Etot
[E] + [ES]
What does the line weaver - burn plot do
Converts curved rate graphs into straight line so Kmax and Km can be calculated
What is the equation for the line weaver burk plot
1/V = Km x1/Vmax[S] + 1/Vmax
In extreme lock and key method what is the effect on the reaction
ES decreases
No stabilising effect on ESt so increases
Vmax and Km decrease
What’s the effect of the extreme induced fit model
No reliance on E + S binding
Vmax higher
As E doesn’t bind well more [S] needed to reach 1/2 Vmax so Km increases
What Kcat
Number of moles of product formed per mol of enzyme per second
What the equation for Kcat
Kcat = Vmax / Etot
what is negative feedback inhibition
high concentrations of product cause inhibition of enzymes
CTP regulates binding in what way
When CTP binds the quaternary structure changes shape causing active site to not be formed preventing the formation of enzyme substrate complexes
stabilizes the t site prevening transition into the rstate that will more readily accept substrate
Mechanisms of Enzyme Catalysis
- Covalent catalysis (serine proteases)* Acid-base catalysis (serine proteases and carbonic anhydrase)* Metal ion catalysis (carbonic anhydrase)* Proximity and orientation effects (serine proteases, CA, ATCase)* Electrostatic catalysis (serine proteases)* Preferential binding to transition state (transition state stabilization) (serine proteases
advantages of using enzymes in biotechnology
stereospecific
Regiospecificity
Substrate specificity
Easy to manipulate / engineer biologicalsystems to alter their properties
Natural Biodiversity
Reproducibility / cost
disadvantages of enzymes in biotech
limited temp range
limited pH range
sensitive to organic solvents
ways around the disadvantages of enzymes in biotech
the use of extremophile enzymes
pros and cons of using enzymes in vitro
Pro:* Optimise and control simple reaction conditions (pH, conc, temp.)* Simple product purification (less contaminants than cells)* No biological contamination problems
Cons:* Enzyme must be isolated from a cell* Pure proteins not stable indefinitely* Some reactions require specific inputs (e.g. ATP / NADPH driven reactions)
pros and cons of using enzyme in vivo
Pro:* Cheap and easy to grow many microorganisms* Link multiple reactions* Controlled intracellular environment ideal for enzyme function
Cons:* Biological contamination problematic in drug formulation* Potential problems transporting enzyme substrates into cells* Unwanted downstream processing of product by organism
how would you manipulate enzyme protein sequence for better performance
Use site-directed mutagenesis to alter sequence of gene encoding an enzyme
