L11 - enzyme substrate reactions Flashcards
Why bother studying enzyme kinetics?
- scientific reasons
- clinical diagnostics
- drug development
what are the scientific reasons?
- enzyme kinetics (how fast reactions occur)
= important for understanding biological (disease) processes
= describes absorbance of drugs into cells by active transport
what is the clinical diagnostics importance?
clinical tests depend on measuring the activity of enzymes
what are the reasons behind the study of enzyme kinetics in drug development?
- 50% of drugs used are enzyme inhibitors
- kinetics tells us about the mechanisms of an enzyme reaction
= allows determination of drug potency
what factors affect enzyme activity?
- pH (inappropriate pH unfolds proteins)
- denaturing reagents
- temperature (high temp = high rate, until thermal denaturation)
- activity is proportional to conc of enzyme
- substrate conc
- presence of inhibitors
how do enzymes work?
- substrate binds to active site on enzyme
- forms enzyme substrate complex
- goes through catalysis
- forms enzyme product complex
- product unbinds, enzyme released
What is enzyme conc like compared to substrates? and why?
[E] «_space;[S]
- because enzymes are catalysts
What do simple models assume?
- 1 molecule of 1 substrate binds to enzyme
- ES complex formed (rate-limiting step)
- enzyme converts substrate to products
- product binds weakly to enzyme
What are the units in the Michaelis-Menten curve?
v = rate
[S] = substrate conc
Vmax = rate when all enzyme active sites are occupied (saturated)
Km = [S] when v = 1/2Vmax
What is the equation for rate in the Michaelis-Menten curve?
v = Vmax[S]/Km + [S]
How to know when the reaction is first order with the MM eqn?
at low [S], rate (v) increases with increasing [S]
How to know when the reactions is zero order with the MM eqn?
at high [S], rate (v) doesn’t increase with further increases in [S]
what are the assumptions of steady-state model?
- [E] «_space;[S]
- [S] remains constant
- [ES] remains the same
- product weakly binds weakly to enzyme
- rate of conversion of product to substrate is negligible (no backward reaction)
What is method 1 of measuring Vmax and Km?
Michaelis-Menten plot
- directly plot v against [S]
- can be difficult to decide when Vmax is reached
= requires a computer programme
What is method 2 of measuring Vmax and Km?
Lineweaver-Burk plot
- plot 1/v against 1/[S]
- y int = 1/Vmax, x int = -1/Km
- higher precision, lower accuracy
- errors are not equal at all points
