The Lineweaver-Burk

To analyze the activity of alkaline phosphatase at different substrate concentrations, a continuous assay was conducted, where the absorbance (at 400 nm) of the enzyme-substrate solution was monitored and recorded over the course of 70 seconds at 10 second increments. This assay was used to determine the rate at which alkaline phosphatase can dephosphorylate p-nitrophenyl phosphate to p-nitrophenol, which then dissociates to phenolate ion, which causes the solution to turn yellow in a solution at a pH of 9.0 (assumed optimum pH) (McCollam-Guilani, p.71). The color change causes the change in absorbance measured by the spectrophotometer. The rate (V0) was calculated by plotting the absorbance against time, where the slope of the line indicates

The Vmax values, as determined from the Lineweaver-Burk plot, for the uninhibited, half uninhibited, and inhibited enzymes were, 0.3647, 0.1262, and 0.3087 μmol/min respectively. The non-linear regression V¬max¬ values for the same enzyme were 0.3343 (9.09% error as compared to Lineweaver-Burk plot), 0.1264 (0.16% error), and 0.2694 μmol/min (14.6% error) respectively. The differences in the values are due to the presence of error introduced by a Lineweaver-Burk plot, where data points at higher and lower substrate concentrations are weighed differently (Tymoczko, p.115). This error is the reason why a Michaelis-Menten plot is preferred. The Vmax for the inhibited reaction was lower than the Vmax for the normal uninhibited reaction, because Orthophosphate inhibitor prevented the enzyme from dephosphorylating the p-nitrophenyl, decreasing the maximal rate of reaction. The Vmax¬ for the reaction where only half of the original enzyme was present was the lowest, because there was less enzyme available. In the Michaelis-Menten plot, the line for the half enzyme uninhibited reaction is the furthest to the right and the curve does not extend as far up, because there is less available enzyme that can be saturated by the

116). Catalytic efficiency deals with how efficiently an enzyme can encounter the substrate and cause the reaction to proceed to products. The Kcat’s for the uninhibited, half uninhibited, and inhibited enzymes were calculated to be 1528.2, 1155.7, and 1231.5 min-1 and the catalytic efficiencies were calculated to be 10161.1, 13974.1, and 3587.4 min¬¬-1 mM-1. The turnover number (K¬¬cat) for the half uninhibited and inhibited reactions were smaller than that of the normal uninhibited alkaline phosphatase, because there was less functional enzyme present, which prohibited the formation of more product and therefore a less intense color change. The catalytic efficiency of the half uninhibited enzyme was higher than the normal uninhibited enzyme, because the likelihood of the enzyme encountering the enzyme was higher, since the ratio of enzyme to substrate decreased. The catalytic efficiency for the inhibited enzyme was lower, because the concentration of enzyme stayed the same and the inhibitor blocked the