Question

In the substrate with inhibitor assay, the inhibitor used was Monopotassium Phosphate, (KH₂PO₄). Please describe the type of inhibition provided by this inhibitor and your reasoning for your determination.

Answer 1

Monopotassium phosphate (1mM) is an inhibitor of acid phosphatase enzyme. It is the inorganic phosphate in KH2PO4, which is the inhibitor. Monopotassium phosphate is a competitive inhibitor of the enzymes. It increases the Km but not the Vmax. Vmax remains constant. This compound binds to the active site of the enzyme. This induces a conformational change in the active site, such that the enzyme no long binds to the substrate. However, the reaction velocity is not affected.

Alkaline phosphatase breaks down paranitrophenol phosphate to para nitrophenol, which is yellow color compound, and phosphate. In the assay, different concentration of Substrate para-nitrophenol are added to enzyme and incubated at 37⁰C. Enzyme substrate reaction can be terminated by potassium hydroxide. Absorbance is analyzed at 405 nm. In separate set of tubes, the inhibitor is added and the absorbance measured.