Lab 4: Enzymes, Seawater plating & Vibrio Colony Streak

Below is an image shown from the lab lecture:

Which of the following substrates has the highest EC₅₀?

 

Substrate A

 

Substrate B

 

Unable to determine from this graph

 

Neither; both have the same EC50

Unable to determine from this graph

**REACTION RATE cannot determine EC50. It can determine Km.

What is the purpose of graphing the reciprocal of reaction velocity and substrate concentration (i.e. the Lineweaver-Burk plot?

 

It makes all the negative values become positive

 

You can calculate the amount of product formed for any given enzyme concentration

 

You can determine the activation energy for the reaction

 

V_max can be calculated from the y-intercept

V_max can be calculated from the y-intercept

Based on the Lineweaver-Burk plot below, what type of inhibitor was used? 

 

Mixed inhibitor

 

Non-competitive inhibitor

 

Competitive inhibitor

 

Unable to determine

Competitive inhibitor

This is a standard absorption curve for ONP.  You conduct the enzyme assay described in Lab 4 to produce ONP, and you measure an absorbance of 0.625 at 420 nm. Given the equation of the regression line in the standard curve, calculate the amount (μmoles) of ONP product generated in the assay tube for this absorbance value (420 nm wavelength). Note that you are calculating μmoles of ONP produced in a 4 mL assay volume, not simply the μM concentration. Write the number in the space provided (as a number only). Round to the nearest hundredths--your answer should have three digits after the decimal. 

0.798

You collect absorbance data for the β-galactosidase assay in Lab 4. You enter your data into the Enzyme Data Analysis Excel file and calculate the μmoles of ONP present at each time point for the reaction that started with 3.2 mM of ONPG. The μmoles ONP data for time points 0.5 - 2 minutes is shown in the table below. You decide to use time points 0.5 min and 1.5 min to calculate the initial velocity.

 

 

What is the initial reaction velocity in μmoles of ONP/min? Write the numerical value in the space provided (include all the numbers after the decimal point).

0.182

For the following Michaelis-Menten graph, what is the K_m (in mM)? Write your answer in the box below (as a number only). You only need to include one number after the decimal.

0.5

The plot shown below is a ______. 

If Line A represents enzyme activity without an inhibitor present, Line B was represents enzyme activity with a _______ inhibitor present.   

Lineweaver-Burk plot

competitive inhibitor