BL483 BASIC BIOCHEMICAL TECHNIQUES -- Lecture 3 - Enzyme Kinetics -- Expt 2 Part B
2. Substrate Saturation of the Enzyme
The definition of a catalyst is something which accelerates the rate of a reaction without being changed itself. For a biocatalyst or enzyme to be most effective, it should be able to do its job with a much lower concentration of itself as compared to the substrate(s) it processes. This leads to the concept that there are a limited number of catalytic sites in a solution containing enzyme and substrate(s): [E] much less than [S]. Consequently, the enzyme will become "saturated" with substrate and the initial velocity (v) of the enzyme catalyzed reaction will react a limiting value called the maximum velocity (Vmax). This is most easily observed by plotting initial velocity versus substrate concentration.
Graph 1. Vo vs. [S] plot.
This is response of the enzyme catalyzed reaction can be described by the Michaelis-Menton equation:
Figure 3. Michaelis-Menton Equation.
This equation defines the shape of a square hyperbola, which is the shape shown for the plot of Vo vs. [S]. The constants -- Vmax and Km --- can be obtained from a set of experimental data where Vo is measured at different [S]. You are doing this experiment this week in lab for the acid phosphatase. And while you need to make a plot of Vo vs. [S] and turn it in as part of your lab report to establish that the phosphatase is obeying the Michaelis-Menton equation, it is best to use other methods to obtain values for Vmax and Km.
But before we get to that stuff, let us make a couple of definitions a little clearer. Vmax is the asymptote of the graph which Vo approaches at very high [S]. The units of Vmax are the same as those of Vo which should be in terms of the quantity of product formed per unit time (i.e. for the acid phosphatase Vo and Vmax should be in µmol or nmol of p-Nitrophenolate formed/min or hr). Operationally, the Km is defined as the substrate concentration which gives 0.5 Vmax (verify this for yourself by substituting [S] = Km in the Michaelis-Menton equation shown above). So Km values are in the same units as substrate concentration (i.e. for the acid phosphatase if [pNPP] is in µM then Km must also be in µM). However, more importantly, the Km is a measure of the strength of the Michaelis complex between enzyme and substrate. Let us make a clear distinction between the binding constant of substrate to the enzyme (Ks) and the Michaelis Constant (Km) -- it is often stated that the Km measures the "affinity" of the enzyme for its substrate. But this is only true in special cases because the Km also includes a measure of how well the enzyme catalyzes the formation of product after the enzyme substrate complex has formed.
As it turns out the ratio of Vmax/Km is the best way to compare enzymes. The "V/K" constant, as it is sometimes called, is a measure of the "efficiency of catalysis" -- if the V/K is large then the enzyme is very efficient. However, there is an upper limit to efficiency which is determined by how fast the substrate can diffuse to the enzyme in solution. Some enzymes have attained this limit over evolutionary time, but most have not and are limited by chemistry that takes place during catalysis.