Enzyme Specific Activity Calculation
The specific activity of an enzyme is a measure of its catalytic efficiency per unit of protein. It is calculated by dividing the enzyme's activity by the total protein concentration.
Given Data:
- Protein Concentration: 10 mg $mL^{-1}$
- Sample Volume: 10 $\mu L$
- Activity in Sample: 5 $\mu mol$ $min^{-1}$
Calculating Protein Mass in Sample
First, determine the mass of protein present in the 10 $\mu L$ sample.
- Convert the sample volume from $\mu L$ to $mL$: $ 10 \, \mu L = 10 \times 10^{-3} \, mL = 0.01 \, mL $
- Calculate the protein mass using the concentration: $ \text{Protein Mass} = \text{Concentration} \times \text{Volume} $ $ \text{Protein Mass} = (10 \, \text{mg} \, mL^{-1}) \times (0.01 \, mL) $ $ \text{Protein Mass} = 0.1 \, \text{mg} $
Determining Specific Activity
Now, calculate the specific activity using the measured activity and the calculated protein mass.
The formula for specific activity is:
$ \text{Specific Activity} = \frac{\text{Enzyme Activity}}{\text{Total Protein Mass}} $Substitute the given values:
$ \text{Specific Activity} = \frac{5 \, \mu mol \, min^{-1}}{0.1 \, mg} $ $ \text{Specific Activity} = 50 \, \mu mol \, min^{-1} \, mg^{-1} $Therefore, the specific activity of the crude enzyme preparation is 50 units $mg^{-1}$ (where units are $\mu mol \, min^{-1}$).
Important Questions from Enzyme Kinetics and Michaelis Menten Equation
- An enzyme (E) catalyzes the biochemical reaction $A \rightarrow B$ with $k_{cat}$ equal to $500 s^{-1}$. If the initial reaction velocity ($V_0$) is $10 \mu M.s^{-1}$ at the total enzyme concentration $[E_t]$ of 30 nM and substrate concentration $[A]$ of $40 \mu M$, the value of $K_m$ (in $\mu M$) is ________
Within the Michaelis-Menten framework, the ratio of $v_0/V_{max}$
when $[S] = 20 \times K_m$ is _________.
(Round off to two decimal places)
The enzyme $\alpha$-amylase used in starch hydrolysis has an affinity constant ($K_m$) value of $0.005$ M. To achieve one-fourth of the maximum rate of hydrolysis, the required starch concentration in mM (rounded off to two decimal places) is____.
An enzymatic reaction exhibits Michaelis-Menten kinetics. For this reaction, on doubling the concentration of enzyme while maintaining [S] >> [$E_o$],
- In an assay of the type II dehydroquinase of molecular mass 18 kDa, it is found that the $V_{max}$ of the enzyme is $0.0134 \ \mu mol.min^{-1}$ when $1.8 \ \mu g$ enzyme is added to the assay mixture. If the $K_m$ for the substrate is $25 \ \mu M$, the $k_{cat}/K_m$ ratio will be ____________________ $\times 10^4 \ M^{-1}.s^{-1}$.
