Question

-A scientist is studying a mutant strain of bacteria that cannot synthesize isoleucine. After placing them in different mediums with different intermediates, she places some bacterial cells in a medium with Intermediate C and they survive and produce isoleucine. Please state which enzymes in this bacteria’s metabolic pathway are functional and which ones are not.

-In a wild type strain of bacteria, she notices that when isoleucine accumulates its production starts slowing down, what type of regulation is this? Where does the isoleucine bind to?

-In a different strain of bacteria, when isoleucine accumulates, the protein levels of Enzyme 1 decrease, what is ONE way the bacterial cell can achieve this? (be specific)

- What is one-way bacteria can increase the levels of Enzyme 1?

Initial substrate (threonine) Active site available -Threonine in active site LEn yme 1 (threonine deaminase) Isoleucine used up by cell Intermediate A En yme 2 Intermediate B En yme 3 Intermediatec En yme 4 Intermediate D En yme 5 End product (isoleucine)

Answer 1

Answer: Amino acid isoleucine is formed in bacteria in a 5-step reaction from threonine The reaction may be summarized as follows: Threonine $\xrightarrow[]{Threonine deaminase}$ $\alpha$-Ketoglutaric acid (intermediate A) $\xrightarrow[]{enzyme 2}$ $\alpha$-Acetohydroxybutyrate (intermediate B) $\xrightarrow[]{enzyme 3}$ $\alpha$-$\beta$ Dihydroxybutyrate (intermediate C) $\xrightarrow[]{enzyme 4}$ $\alpha$-keto-$\beta$-Methylvalerate (intermediate D) $\xrightarrow[]{enzyme 5}$ Isoleucine.

Here the mutant strain of bacteria cannot synthesize isoleucine, but after placing in a medium with intermediates C, they survive and produce isoleucine. So the enzyme 1, enzyme 2 and enzyme 3 are non functional while, enzyme 4 and enzyme 5 are functional. So the functional enzyme are: enzyme 4$\rightarrow$ Dihydroxy dehydratase; enzyme 5 $\rightarrow$ isoleucine transaminase. The non-functional enzymes are: enzyme 1 $\rightarrow$ Threonine deaminase; enzyme 2 $\rightarrow$ acetohydroxy acid synthase; enzyme 3 $\rightarrow$ Acetohydroxy acid isomeroreductase.

Answer: The accumulation of isoleucine beyond a threshold value, reduces its further production. This is an example of allosteric regulation where some specific low molecular weight substrance, such as the product(s) acts as the inhibitor by binding with a specific site of the enzyme different from its substrate binding site. The binding of the regulatory molecule can either enhance the activity of the enzyme (allosteric activation) or inhibit the activity of the enzyme (allosteric inhibition). Likewise the accumulation of isoleucine further stops its production by the inhibition of the enzyme activity.

The end product, isoleucine binds to a site other than the substrate binding site known as allosteric site.

Answer: When isoleucine accumulates beyond a threshold value, its further production stops because it inhibits the activity of enzyme 1, threonine deaminase. This is known as feedback inhibition and is allosterically regulated.

The activity of enzymes may be increased or decreased by covalent modification. It means, either addition of a group to the enzyme protein by a covalent bond, or removal of a group by cleaving a covalent bond. Here the bacterial cell can able to decrease the level of enzyme 1, by dephosphorylation, i.e, removal of phosphate group by hydrolysis which cleave ester bond that switched off the enzyme activity.Thus the biosynthetic machinery of isoleucine pathway synthesis is inhibited. The enzyme 1, threonine deaminase is downregulated by isoleucine and leucine. So the addition of isoleucine and leucine results in decreased levels of enzyme 1.

Answer: Contrarily, phosphorylation increases the levels of enzyme 1. The phosphate group may be attached to threonine residues which activates the intracellular signaling by switching on the enzyme activity. Thus a downstream cascades is initiated resulting in the higher levels of the desired enzyme. THreonine deaminase's activity is upregulated by valine, so the addition of valine increases the levels of threonine deaminase.