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BCMB3100 with Paula Lemons and Takahiro Ito...

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Introduction to Biochemistry and Molecu lar Biology Molecular BCMB 3100 31276

Case Study

Amino Acid Metabolism

Take ake-Home -Home Poi Point nt Like glycolysis and gluconeogenesis, other metabolic pathways are governed by principles we learned in Unit 2. For example, enzymes in the pathways are highly regulated, and enzyme-mediated regulatory mechanisms allow these pathways to be sensitive and responsive to the needs of the organism. These pathways also illustrate how metabolism is linked to the energy status of cells, consuming or generating energy in the form of molecules like ATP and NADH. Ultimately, these metabolic pathways are integrated and connected. For example, the substrates and products of the citric acid cycle are involved in gluconeogenesis, fatty acid synthesis, amino acid synthesis, and other pathways.

Learning Objective Objectivess 1. 2. 3. 4. 5. 6. 7. 8.

Describe a transamination reaction and draw the structures of substrates and products. Describe oxidative deamination of glutamate to form NH4+. Describe the steps of the urea cycle and state the source of the nitrogen in urea. List the seven molecules that are produced from the carbon skeletons of amino acids after deamination. Differentiate between ketogenic and glucogenic amino acids. Summarize the process of amino acid synthesis. Describe cumulative feedback inhibition and how it is used to regulate amino acid synthesis . Manipulate a biosynthetic pathway to optimize production of a specific amino acid.

Introduction “We turnover about 400 grams and eliminate about 100 grams of protein a day, hence the notion that we should eat about 3-4 oz. of protein a day. This corresponds to about 5 g of nitrogen. If we resupply the lost nitrogen we are said to be in neutral nitrogen balance. When we are losing protein we are in a negative nitrogen balance and when we gain protein we are in a positive nitrogen balance. Generally this corresponds to losing and building muscle mass. ” – Loertscher and Minderhout, Foundations of Biochemistry, 3rd ed , p. 139. This case examines the synthesis and degradation of amino acids. It also covers the metabolism of nitrogencontaining molecules, from amino acids to ammonium ion to urea.

Adapted by Cheryl Sensibaugh (Fall 2017) from a case written by Paula Lemons.

Pa Part rt I – Amino Acid Degr Degradation adation 1. A ketoacid contains a ketone group and a carboxylic acid group. An -ketoacid has the ketone group adjacent to the carboxylic acid group. In contrast, a -ketoacid has the ketone group at the second carbon from the carboxylic acid. a. Referring to Figure 19.6, list the citric acid cycle intermediates that are -keto acids. Alpha ketoglutarate and oxalcoacetate b. In your own words, describe the general role of an -keto acid in a transamination reaction and the ultimate goal that makes a transamination reaction necessary. Transamination transfers an amino group to a ketoacid to form new amino acids. Alpha ketoglutarate acts as the predominant amino-group acceptor and produces glutamate as the new amino acid.

2. The complete removal of nitrogen from an amino acid requires two reactions. First, transamination reactions collect amino groups from many different amino acids in the form of glutamate. Consider the specific example of the transamination of aspartate: aspartate + -ketoglutarate  oxaloacetate + glutamate  Draw the chemical structures of the substrates and products.  Label the substrates and products.  Label the name of the enzyme. Then, oxidative deamination removes the nitrogen from glutamate to form NH4+ (an ammonium ion).    

On the drawing you started above, add a reaction arrow to show oxidative deamination. Label the name of the enzyme. Label the additional substrates and products. Insert a photo of your drawing below.

3. How does a low energy charge affect oxidative deamination? What is the benefit of the reduced product? Lowering the energy charge (more ADP and GDP) accelerates the oxidation of amino acids. A reduced product

4. Ammonium is toxic and must be converted into a molecule that can be safely transported for excretion. Draw a diagram of a cell showing the reactions of the urea cycle in the appropriate subcellular locations. Start with ammonium and end with urea.  Label the cell type.  Label the molecules by name; chemical structures are not necessary.  Label the name of the enzyme for which free ammonium is a substrate.  Circle the names of the -amino acids.  Put a star beside the -amino acids of the urea cycle that can be used to build proteins.  Insert a photo of your drawing below.

5. Which enzyme involved in nitrogen removal is regulated? What makes this step an ideal one to regulate? Glutamine synthetase; glutamine and glutamate can be synthesized from alpha ketoglutarate, an intermediate from the TCA cycle.

6. What happens to the carbon skeletons of amino acids after the -amino group is removed? Carbon skeleton remains as a carboxylic acid (usually a 2-ketoacid) that can be used for synthetic purposes or be degraded. Some can be converted to glucose by gluconeogenesis and some (ketogenic) can be converted to produce acetyl coA.

7. In your own words, summarize the differences between glucogenic and ketogenic amino acids.

A ketogenic amino acid is an amino acid that can be degraded directly into acetyl-CoA, which is the precursor of ketone bodies. This is in contrast to the glucogenic amino acids, which are converted into glucose.

Pa Part rt II – Amino Acid Sy Synthesis nthesis 8. Building an amino acid can be thought of as requiring two building blocks: a source of nitrogen, and a carbon skeleton. Yet atmostpheric nitrogen, N2, is essentially useless to most organisms. Summarize amino acid synthesis by answering the questions below. a. What is the name of the process that converts atmostpheric nitrogen to a biologically useful form? Nitrogen fixation b. What is one type of organism that is able to carry out this conversion? Nitrogen fixing bacteria/ protozoa c.

What is the name and chemical formula of the uncharged nitrogenous molecule that is produced? Ammonia- NH3

d. What happens spontaneously to that uncharged nitrogenous molecule? It attracts a proton to form NH4+ e. What enzyme catalyzes the addition of that nitrogen source onto a carbon skeleton? The nitrogenase enzyme f

serves as a substrate for that enzyme?

g

ed?

9. A r f a d a

f amino acids is tightly regulated. We will consider the anism Corynebacterium glutamicum. Researchers published the L-threonine or pyruvate into other amino acids (Elisakova et st of the enzymes are capable of catalyzing reactions using . In other words, depending on the starting materials, different of the figure, multiple arrows indicate multiple enzymes. Abbr.

Enzyme

TD

Threonine dehydratase

AHAS

Acetohydroxy acid synthase

AHAIR DHAD TA

Acetohydroxy acid isomeroreductase Dihydroxyacid dehydratase Transaminase

Gene

ilvA ilvB (catalytic subunit) ilvN (regulatory subunit) ilvC ilvD ilvE

a. What are the two reactions that AHAS is capable of catalyzing? 2-ketobutyrate and pyruvate or pyruvate and pyruvate (ilVB and ilvn) b. If AHAS forms 2-aceto-2-hydroxy-butyrate, which amino acid(s) is/are ultimately produced? l-isoleucine c.

If AHAS forms 2-acetolactate, which amino acid(s) is/are ultimately produced? l-valine and l-isoleucine

d. Describe what happens after 2-ketoisovalerate is formed. TA forms L-valine; also production of l-leucine

10. C. glutamicum employs cumulative feedback inhibition of AHAS by valine, isoleucine, and leucine. a. In your own words, define cumulative feedback inhibition. In cumulative inhibition, each inhibitor can reduce the activity of the enzyme, even when other inhibitors are bound at saturating levels. b. Predict the minimum number of allosteric binding sites on AHAS. 3 c.

Explain your reasoning for your prediction.

Is only inhibited by 3 binding sites, so there can only be 3 allosteric binding sites

11. AHAS from C. glutamicum is comprised of a catalytic subunit and a regulatory subunit, encoded by the ilvB and ilvN genes, respectively. Elisakova and colleagues tested mutations in the ilvN gene to determine whether mutation might prevent cumulative feedback inhibition. If successful, the mutation would be said to confer resistance to cumulative feedback inhibition. Their data are shown in the table below. Note that experimental error was quantified by the standard deviations of at least three independent measurements.

Relative activity (%) in presence of 10 mM amino acida

Mutation on plasmid

None

Valine

Isoleucine

Leucine

None M8 M11 M13

100 100 100 100

52 112 32 95

57 112 61 108

65 105 82 98

a

The values are averages of at least three independent measurements; the standard deviations were within 18%. a. If a result is within the standard deviation, are you confident concluding that the activity of AHAS changed? Explain your reasoning. If a result is within the standard deviation, the activity of AHAS increases because product is catalyzed by the enzyme. b. For the M8 ilvN mutant, state the result of the activity assay and the conclusion that can be drawn from that result. Specifically, how does activity of the mutant compare to the wild type control, and is the mutant resistant to cumulative feedback inhibition? If so, which amino acid(s) no longer impact AHAS activity? Leucine is no longer impacted by AHAS activity. Not resistant to cumulative feedback inhibition because sit is not active. c.

For the M11 ilvN mutant, state the result of the activity assay and the conclusion that can be drawn from that result. Specifically, how does activity of the mutant compare to the wild type control, and is the mutant resistant to cumulative feedback inhibition? If so, which amino acid(s) no longer impact AHAS activity? Valine is not impacted, m11 is weakest because it has lowest relative activity.

d. For the M13 ilvN mutant, state the result of the activity assay and the conclusion that can be drawn from that result. Specifically, how does activity of the mutant compare to the wild type control, and is the mutant resistant to cumulative feedback inhibition? If so, which amino acid(s) no longer impact AHAS activity? High inhibtion of isoleucine.

12. C. glutamicum is used for commercial production of a number of amino acids, including L-valine. Imagine that the UGA Bioexpression and Fermentation Facility (BFF) searched for a strain of C. glutamicum that would allow even greater production of L-valine. Researchers at the BFF narrowed potential candidates to three strains with various combinations of mutations. The strains contained the following mutations, with  indicating a gene deletion:  Strain 1 – ilvNM13

 Strain 2 – ilvA and panB  Strain 3 – ilvA , panB, and ilvNM13 a. Using the previous figure of amino acid synthesis in C. glutamicum, explain how the ilvNM13 mutation would alter metabolism to impact the production of L-valine. L-valine inhibition increases because mutation does not show feedback inhibition b. Using the previous figure of amino acid synthesis in C. glutamicum, explain how the ilvA deletion would alter metabolism to impact the production of L-valine. Deletion would halt production of v-isoleucine c.

Using the previous figure of amino acid synthesis in C. glutamicum, explain how the panB deletion would alter metabolism to impact the production of L-valine. Valine production increases due to pyruvate production

Upon testing each strain for production of L-valine, researchers obtained the results shown below.

d. Which strain should the UGA BFF use to maximize L-valine production? Strain 3

fate of skeleton backbone of aminoacids after getting degraded: acetyl coa, acetoaacetyl coa, pyruavte, oxaloacetat,e fumarate, succinyl coa, alpha ketoglutarate amino acids cant produce amino acids y less theyre odd creates ketone bodies: acteyl coa, NADH...