To continue the topic about enzyme kinetics, you will find some good pdfs below.
The main points to remember about enzyme kinetics are graphs with various types of inhibition.
Let us examine enzyme kinetics as a function of the concentration of substrate available to the enzyme.
- We set up a series of tubes containing graded concentrations of substrate, [S].
- At time zero, we add a fixed amount of the enzyme preparation.
- Over the next few minutes, we measure the concentration of product formed. If the product absorbs light, we can easily do this in a spectrophotometer.
- Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observe is the initial velocity of Vi.
Plotting Vi as a function of [S], we find that
- At low values of [S], the initial velocity,Vi, rises almost linearly with increasing [S].
- But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).
- The asymptote represents the maximum velocity of the reaction, designated Vmax
- The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km (named after the scientists who developed the study of enzyme kinetics).
Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).
Plotting the reciprocals of the same data points yields a “double-reciprocal” or Lineweaver-Burk plot. This provides a more precise way to determine Vmax and Km.
- Vmax is determined by the point where the line crosses the 1/Vi = 0 axis (so the [S] is infinite).
- Note that the magnitude represented by the data points in this plot decrease from lower left to upper right.
- Km equals Vmax times the slope of line. This is easily determined from the intercept on the X axis.
The effects of enzyme inhibitors
Enzymes can be inhibited
- competitively, when the substrate and inhibitor compete for binding to the same active site or
- noncompetitively, when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency.
The distinction can be determined by plotting enzyme activity with and without the inhibitor present.
In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. So while Vmax can still be reached if sufficient substrate is available, one-half Vmax requires a higher [S] than before and thus Km is larger.
With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so
- enzyme rate (velocity) is reduced for all values of [S], including
- Vmax and one-half Vmax but
- Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged.
This Lineweaver-Burk plot displays these results.
So let’s proceed to more comprehensive stuff
One more tutorial.
Big thanks to http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html, http://www.columbia.edu/itc/chemistry/chem-c2407/hw/ENZYME_KINETICS.pdf and www.glutxi.umassmed.edu/grad/GradKinetics.pdf.