Question

How would diisopropylphosphofluoridate (DIPF; Section 11-5A) affect the apparent ${\mathbf{K}}_{\mathbf{M}}$ and ${\mathbf{V}}_{\mathbf{max}}$ of a sample of chymotrypsin?

Short answer

DIPF does not affect ${\mathrm{K}}_{\mathrm{M}}$because the uninhibited chymotrypsin will bind the substrate to its active site normally. However, ${\mathrm{V}}_{\max }$ decreases because DIPF decreases the total enzyme concentration when it binds irreversibly with chymotrypsin.

Step-by-step solution

Diisopropylphosphofluoridate

Diisopropylphosphofluoridate is a type of inhibitor that inhibits serine proteases and similar enzymes. It helps to detect Ser in the active site of serine proteases. Thus, the DIPF reaction with thechymotrypsin helps identify the enzyme's active site.

Effect of DIFP on apparent Vmax of chymotrypsin

The total enzyme concentration and maximum velocity are directly proportional, as can be seen from the expression.

$$\begin{gathered} {\mathrm{k}}_{\mathrm{cat}}=\frac{{\mathrm{V}}_{\max }}{{\left[\mathrm{E}\right]}_{\mathrm{T}}} \\ {\mathrm{V}}_{\max }={\mathrm{k}}_{\mathrm{cat}}\times {\left[\mathrm{E}\right]}_{\mathrm{T}} \end{gathered}$$

The active site of the enzyme is permanently inactivated by DIPF. As a result of the activity of DIPF, the overall enzyme concentration is lowered, lowering the maximum velocity.

Effect of DIFP on apparent  of chymotrypsin

The K_M of the enzyme is unaffected because the enzymes that are not inhibited by DIPF bind to the substrate normally.