Question

Some bacteria make an enzyme called β-galactosidase that catalyzes the breakdown of the polysaccharide β-galactoside into monomers of galactose. A population of bacteria were found with an amino acid mutation within the active site of β-galactosidase protein, and these bacteria were unable to breakdown β-galactoside polymers. A) How would you identify a population of bacteria with mutant β.galac tosidase in terms of the relative concentration of β-galactoside and galactose in their cells? B.) Explain how the amino acid mutation could have affected enzyme function. Be specific and use correct terminology (enzyme active site, substrate, etc ) HTML Editor Paragraph

Answer 1

A. A population of mutant bacteria which is unable to metabolize lactose can be found by comparing the lactose utilization between WT and mutant lines are various time points. The amount of lactose remaining in the culture media can be measured by simple biochemistry tests and more the amount of lactose remaining compared to WT, should tell us about the population of bacteria unable to utilize lactose.  

With respect to the amount of galactose present inside the cells, we can do this by a simple protocol of blue-white screening where the cells can be plated on a media containing X-gal. Upon synthesis of Beta-galactosidase enzyme which would happen only if the cells are able to transport lactose and break it down, would result in blue coloration of the colonies and cells which are unable to perform this step would result in white color colonies. Based on the color difference, we can assess whether a given population of cells are capable of lactose metabolism or not.

B. The amino acid mutation could have resulted in change in secondary or tertiary structures of the protein that render it unable to catalyze the breakdown of lactose. On the other hand, mutations in the active site may render the protein's ability to bind lactose impaired or might hinder the enzyme's ability to initiate the catalytic breakdown but substrate binding would still be unaffected.