What are enzymes and what is their function? How can they work on the substrate? What does catalytic activity depend on?
- biological catalysts which can speed up the rate of biochemical reactions
- turn substrate into product
- catalytic activity depends on the integrity of the active site
What are the characteristics of enzymes? List out 5 characteristics.
- highly specific –> catalyse a single reaction
- speed up reaction in either direction –> reversible
- protein in nature –> can be denatured at high temperature or extreme pH value –> disruption of active site and loss of function
- remains unchanged after reaction –> small amount is enough
- may require the presence of other substances in order to function (eg. cofactors/ activators)
What can enzyme cofactors be classified into? How do they bind to enzymes? What is the word equation for the binding?
- inorganic atoms (eg. Fe2+, Mg2+, Zn2+) and organic molecules (=coenzymes)
- most cofactors bind loosely to the protein portion of enzyme (apoenzyme)
- apoenzyme (=protein enzyme) + cofactors = holoenzyme (=completely active enzyme)
What is the equation of the binding of enzyme and substrate? How do enzymes speed up biochemical reactions? What won’t be changed by enzymes? What does it indicate if it’s negative?
- E (active site of enzyme) + S (substrate molecule) –> ES (enzyme-substrate complex) –> EP (enzyme-product complex) –> E (enzyme molecule) + P (product)
- speed up by lowering the activation energy (=energy barrier)
- do not change ΔG (free energy difference between the reactant and product)
- -veΔG –> energy-releasing reaction –> favours product formation as reaction moves to a lower energy state –> products are more stable
How does the specificity of enzyme (lock) and its substrate (key) come from? What will happen during complex formation?
- comes from the complementary shapes
- substrate fits exactly with the active site of enzyme
Why is there an induced-fit model? What will happen during this model?
- substrate and enzyme do not have a perfect complementary structure
- active site forms a complementary shape to the substrate only after substrate binding –> induced fit
What is enzyme kinetics? How is the initial rate different in low and high [S]?
- the study of how the enzymatic reaction changes according to experimental parameters
- at low [S], initial rate (V0) increases linearly with increasing [S]
- at high [S], increase in V0 slows down and eventually reaches a plateau –> maximum velocity (Vmax)
What is the Michaelis-Menten equation? What situation will there be special in Km and [S]?
- refer to notes p.2
- km = [S] when V0=1/2 Vmax
What is V0 when low and high [S], with reference to the Michaelis-Menten equation?
- At low [S], km»[S] so km + [S] = km –> V0 = Vmax/km –> V0 increases linearly with [S] at a slope of Vmax/km
- At high [S], [S]»km so km + [S] = [S] –> V0 = Vmax –> Vmax is observed at high [S] as all active sites are being used up by substrate molecules
What is Lineweaver-Burk equation?
- taking reciprocal of the Michaelis-Menten equation
- refer to notes p.2
Why double-reciprocal plot is useful?
- a plot of V0 versus [S] gives a hyperbolic curve –> Vmax is achieved at infinite [S] –> impossible to accurately determine Vmax and km
- when determining Vmax or km, measure V0 at different concentrations and then plot –> give a straight line (slope=km/Vmax[S]; x=-1/km; y=1/Vmax –> a more accurate Vmax
What factors will affect the rate of enzymatic reactions?
- enzyme concentration
=> enzymatic rate is directly proportional to the enzyme concentration under suitable conditions, provided that an excess of free substrate molecules is present - pH
=> enzymes have optimal activity at specific pH
=> pH lower or higher than the optimal pH will decrease the enzyme activity - temperature
=> Q10=reaction velocity at T+10/reaction velocity at T = 2 –> a rise of 10 in temperature will double the reaction rate
=> increased temperature will increase the kinetic energy of substrate –> more collisions of the substrate with the active site –> more enzyme-substrate complex and product formed
=> in human, most enzymes have optimal temperature at 37, if higher than the optimal temperature –> lower rate due to alteration of active site - enzyme inhibitors
=> reduce enzymatic reaction rate
=> 2 types: reversible and irreversible
What are the three characteristics of reversible inhibitors? What are they further divided into? And how do these inhibitors work respectively?
- effect is temporary
- no permanent change to enzyme
- removal of these inhibitors can restore enzymatic activity
- competitive inhibitors
=> compete with the substrate for active sites -> km increases
=> effect of inhibitors decreases when [S] increases –> Vmax remains unchanged - non-competitive inhibitors
=> bind to an allosteric site –> no competition between inhibitors and substrate for enzyme binding - the substrate can still bind to enzyme but the enzyme cannot catalyse the reaction –> Km remains unchanged and Vmax decreases
How do irreversible inhibitors work?
- destroy a functional group on an enzyme –> formation of covalent bond between the enzyme and inhibitor –> destroy active site –> permanent loss of catalytic activity
What can determine the enzyme inhibition mechanism? How do competitive and non-competitive inhibition affects the unknowns?
- double-reciprocal plot
- competitive inhibition
=> km increases and Vmax unchanged –> slope increases; x-intercept decreases; y-intercept remains unchanged - non-competitive inhibition
=> km unchanged and Vmax decreases –> slope increases; x intercept remains unchanged; y-intercept increases
