Effect of Substrate Concentration on the Reaction Velocity of Alkaline

Phosphatase
Introduction:
In order for many life sustaining chemical reactions to occur rapidly the presence of a catalyst is often required.
In biological systems proteins called enzymes are the catalysts for most reactions. Enzymes differ from normal
proteins in that they contain a specific area of catalytic activity called the active site. It is at the active site that
substrate molecules will bind to the enzyme through weak interactions (Nelson et al. 2008). The interaction
between an enzyme and a substrate will provide an alternative pathway of lower activation energy for the
reaction to occur, increasing the rate of the reaction but not affecting the position of equilibrium of the reaction.
Alkaline phosphatase is a hydrolytic enzyme which catalyzes the removal of phosphate (PO43-) groups from
many types of organic molecules. Alkaline phosphatase is found in many different organisms and has
extensive research and diagnostic use. The increased activity of alkaline phosphatase in the periplasm
compared to the cytoplasm has been used to determine the topology of several membrane proteins (Manoil et
al 1986). Also, increased or decreased activity of the enzyme in the human body can be used in conjunction
with other techniques to diagnose several diseases.
The purpose of this experiment was to determine how substrate concentration affects the catalytic ability of
alkaline phosphatase and, subsequently, to determine the maximum velocity (Vmax) and Michaelis constant
(Km) of the reaction of the enzyme with p-nitrophenyl phosphate.

Method:
Five cuvettes with different concentrations of p-nitrophenyl phosphate were prepared in a slightly basic
environment. A 0.1ml sample of the enzyme alkaline phosphatase was then added to each cuvette and the
cuvettes were run through a spectrophotometer for 2mins. Absorbance was recorded each 15secs. The use of
spectroscopy stems from the difference in light absorbance of p-nitrophenyl phosphate compared to the
product of the enzyme catalyzed reaction, nitrophenol. P-nitrophenyl phosphate does not absorb visible light at
405nm while nitrophenol absorbs light at that wavelength (Hardie 1993). The change in absorption

Results:
The absorbance of the five cuvettes at different time intervals was recorded below (Table 1). Absorbance
change over time was graphed (see appendix) for each cuvette used in the experiment and the initial reaction
velocities were found for the different substrate concentrations (Table 2). The inverses of the substrate
concentrations and the initial reaction velocities are shown in Table 3. From the data in Table 3 a Lineweaver-
Burk plot was created (Figure 1). The x and y intercepts of the Lineweaver-Burk plot were found (Table 4) and,
through the use of the Beer-Lambert law, the Km and Vmax values were determined (work shown below) (Table
5).

Absorbance at 405 nm
Concentration of p-nitrophenyl phosphate

Time (secs) 0.5mM 0.8mM 1.0mM 2.0mM 5mM 10mM

15 0.128 0.123 0.123 0.184 0.314 0.439
30 0.155 0.161 0.168 0.245 0.399 0.523
45 0.183 0.199 0.214 0.307 0.478 0.615
60 0.209 0.238 0.255 0.366 0.56 0.701
75 0.235 0.273 0.299 0.423 0.645 0.785
90 0.259 0.307 0.338 0.482 0.724 0.87
105 0.283 0.342 0.375 0.537 0.801 0.96
 120 0.305 0.372 0.413 0.589 0.884 1.042

Concentration(mM) of p-nitrophenyl Velocity of reaction (Absorbance

phosphate (S) change/min) (V)
0.5 0.1016
0.8 0.1432
1 0.1658
2 0.2322
5 0.3320
10 0.3455

1/substrate concentration mM-1 1/Velocity

2 9.524
1.25 6.803
1 6.410
0.5 4.717
0.2 3.484
0.1 3.226

Value at the intercept of the X-axis (1/S) -0.891

Value at the intercept of the Y axis (1/V) 2.928

Km Value (mM) 1.122

Vmax mol min-1 18.975