Enzyme Inhibition

1
Enzyme Inhibition
Katherine Gaynor Lady Margaret Hall Department of
Biochemistry Oxford University

• Aim
• To find out if inorganic phosphate (Pi)
  competitively inhibits alkaline phosphate using
  enzyme kinetics.

Null Hypothesis Pi does not inhibit alkaline
phosphate.
pNPP binds to alkaline phosphate and is
hydrolysed to form p-nitrophenyl phosphate, a
chromogenic substrate.
This property allows the amount of p-nitrophenyl
phosphate formed to be measured by reading the
absorption at 405nm.
Km the concentration of substrate at 50
saturation of the enzyme.

• Method
• Incubate pNPP with alkaline phosphate for 10mins
• Measure absorbance at A450
• Carry out reaction again with Pi
• Plot a Lineweaver-Burke plot for each reaction
• Calculate Km and Vmax for each reaction.

Lineweaver-Burke Plot
Vmax turnover rate of the enzyme
Conclusion The results show that the enzyme is
inhibited by Pi so the null hypothesis is
rejected. Pi competively inhibits alkaline
phosphatase. This can be deduced as when
inhibitor is added the Km increases but Vmax does
not change. This indicates that Pi binds to the
active site preventing the pNPP binding as the
saturation decreases whereas the turnover rate
is unaffected, so the enzyme is still binding to
substrates. The reason for this type of
inhibition is that Pi is a similar shaped
molecule to pNPP, so can bind to the active site
of alkaline phosphate, preventing pNPP from
binding.
Take home message Inorganic phosphate competively
inhibits alkaline phosphatase!
Pi