Question

Answer any two and only two of the following.

 a. Provide an overview of purine nucleotide biosynthesis and discuss its regulation.

 b. Describe the urea cycle including its function, energy costs and regulation.

 c. Give an overview of lysine, threonine, methionine, and isoleucine biosynthesis in bacteria and discuss its regulation.

 d. Discuss the transcriptional control of the HMG-CoA reductase gene and over 20 other genes linked to cholesterol and fatty acid metabolism.

 e. Compare and contrast the regulation of pyrimidine biosynthesis in bacteria and mammals.

Answer 1

a. Purines are heterocyclic bases.They are one of three components of nucleotides phosphate esters of a pentose sugar (either ribose or deoxyribose) in which a purine or pyrimidine base is linked to C1 of the sugar and closed ring structures comprised of at least two different kinds of atoms.

Purine biosynthesis occurs in the cytosol of all cells. The purine ring is built up in a series of 11 enzyme catalysed steps. Each enzyme is oligomeric, which means it contains several monomers. Intermediate products that are produced during the reaction are not released. Instead, they are shuttled to the subsequent enzyme along the pathway.

Step one of this pathway generates an important compound, 5-phosphoribosyl-alpha-pyrophosphate (PRPP). This compound is also a precursor in the biosynthesis of pyrimidine nucleotides. It provides the phospho-ribose units of these ribonucleotides.

PRPP is derived from ribose-5-phosphate (R5P) a product of the pentose phosphate pathway. Therefore, purines are built from a series of addition reactions to a sugar.

Purine synthesis yields inosine monophosphate

In the first step of purine biosynthesis, ribose phosphate pyrophosphokinase activates the ribose by reacting it with ATP to form 5-phosphoribosyl-alpha-pyrophosphate (PRPP).

Step 2 is the committed step of purine biosynthesis. In this reaction amidophosphoribosyl transferase catalyses the displacement of PRPP’s pyrophosphate group by glutamine’s amide nitrogen. This reaction is the pathway’s flux-controlling step (the rate at which the biosynthetic pathway outputs product) . Following the remaining 9 steps, the first purine derivative that is synthesised is inosine monophosphate (IMP).IMP is the precursor for the purine nucleotide, adenosine and guanosine monophosphate (AMP and GMP). Each are synthesized in a two-reaction pathway with bifurcates at the level of IMP:

Further phosphate additions to generate diphosphate and triphosphate nucleosides may follow completion of monophosphate synthesis. These reactions are carried out by kinases.

Kinases are so-called due to their property of transferring phosphate groups from a high-energy phosphate molecule to specific substrates. The complete nucleotide triphosphate forms, adenosine and guanosine triphosphate (ATP and GTP) are the recognizable units of RNA and DNA. Therefore, purines are initially formed as ribonucleotides rather than as free bases.

Glycine O=C -o- D-O- ATP AMP Мо? PAPP SHH POH OH OH 0-D-Ribose 5-phosphate NH Mo--O- OH OH Phosphoridosyl pyrophosphate (PRPP)00) More NEN (9)ỘC, Oce ΝΗ Metrony Glutamine Gutamate 0-0-HC H NH HOPP MooH folate не се AH HI А НА 0 PAPP Gramy HIM Am dotransferase ATP ADPP UM н Formyanserase ONH OH OH OH OH S-Phospho-ribosline Glycinamide ribosyi 5.phosphate Formylglycinamide TV) ribosyl-5-phosphate (V) an ATP VI Synthese 1 Asp ATP 000 "ООО ABP -P CO ATP. Mg HO Ring closure H.CH HC HC OOC Fumar OOC RS Aminoimidazole sucoin! carboxamide ribesyl-5-phosphate (IX) x Shotsa VII Carboys ASP Aminoimidazole carboxylateriboylphosphate (VII Vil Syntetise ASD Aminoimidazole ribosyl-5.phosphate RS- Formyiglycinamidine ribosy 5.phosphate (VD Adenylsuccinas NFommy Holate He folate HO HON HN Fomy transforce HNT O=C H H Ang closure IMP Cycohydrolase Inosine monophosphate (IMP) Aminimidazole carboxamide ribosyl-5-phosphate (X) Formimidimidrole carbonide ribosyl-5-phosphate

Purine nucleotide biosynthesis is regulated at several steps

This is crucial to prevent the waste of

(1) energy and nitrogen

(2) to control the total amounts of purine nucleotides available for nucleic acid synthesis

(3) the purine waste product, uric acid are harmful to cells.Excessive uric acid production leads to its deposition in joints causing pain and redness.

IMP synthesis is controlled by the levels of adenine and guanine nucleotides. Additional control is exerted by feedforward activation which is the stimulation of a subsequent enzyme by the preceding substrate. In this situation, the amidophosphoribosyl transferase I step 2 is allosterically stimulated by PRPP, the product of step 1.

The second level of regulation occurs at the branch point below IMP, leading to either AMP or GMP. These end products are each competitive inhibitors of IMP and so, their excessive build-up is prevented.

The metabolic requirement for purine can be fulfilled by biosynthesis in the human body. Without adequate production of purines, or because of abnormal biosynthetic pathways, painful clinical manifestations can arise.

b. Urea cycle

The urea cycle is the metabolic pathway that transforms nitrogen to urea for excretion from the body.

• Nitrogenous excretory products are removed from the body mainly in the urine.
• Ammonia which is very toxic in humans is converted to urea which is nontoxic, very soluble and readily excreted by the kidneys.
• The urea excreted each day by a healthy adult (about 30 g) accounts for about 90% of the nitrogenous excretory products.

Urea is formed in the urea cycle from NH4+, CO2, and the nitrogen of aspartate.The cycle occurs mainly in the liver.

CO + NH4+ Carbamoyl phosphate synthetase / Carbamoyl phosphate Urea Cycle Aspartate Ornithine transcarbamoylase Citrulline Arginosuccinate synthetase Enzymes & Ornithine Arginosuccinate Steps Urea Arginase Arginosuccinase Cytoplasm Mitochondria Rate limiting step H2O Arginine Fumarate

Cytosol and mitochondria of hepatocytes.The Substrates used are NH3 (as derived from oxidative deamination of glutamate) CO₂ ,aspartate ,three ATP.The products formed are Urea, fumarate,H₂O.

Steps in Urea cycle

1.Transport of nitrogen to the liver

Ammonia is very toxic, particularly to the central nervous system.The concentration of ammonia and ammonium ions in the blood is normally very low.

(NH₃ + H+ ↔ NH₄+)

Ammonia travels to the liver from other tissues, mainly in the form of alanine and glutamine.It is released from amino acids in the liver by a series of transamination and deamination reactions.Ammonia is also produced by bacteria in the gut and travels to the liver via the hepatic portal vein.

2. Reactions of the urea cycle

NH₄+ and aspartate provide the nitrogen that is used to produce urea and CO₂ provides the carbon. Ornithine serves as a carrier that is regenerated by the cycle.Carbamoyl phosphate is synthesized in the first reaction from NH_4⁺, CO₂ and two ATP. Inorganic phosphate and two ADP are also produced.

• Enzyme: carbamoyl phosphate synthetase I, which is located in mitochondria and is activated by N-acetylglutamate.Ornithine reacts with carbamoyl phosphate to form citrulline. Inorganic phosphate is released.
• Enzyme: ornithine transcarbamoylase, which is found in mitochondria. The product, citrulline, is transported to the cytosol in exchange for cytoplasmic ornithine.
• Citrulline combines with aspartate to form argininosuccinate in a reaction that is driven by the hydrolysis of ATP to AMP and inorganic pyrophosphate.
• Enzyme: Argininosuccinate synthetase
• Argininosuccinate is cleaved to form arginine and fumarate.
• Enzyme: argininosuccinate lyase. This reaction occurs in the cytosol.
• The carbons of fumarate, which are derived from the aspartate added in reaction 3, can be converted to malate.
• In the fasting state in the liver, malate can be converted to glucose or to oxaloacetate, which is transaminated to regenerate the aspartate required for reaction 3.
• Arginine is cleaved to form urea and regenerate ornithine.
• Enzyme: arginase, which is located primarily in the liver and is inhibited by ornithine.
• Urea passes into the blood and is excreted by the kidneys.
• Ornithine is transported back into the mitochondrion (in exchange for citrulline) where it can be used for another round of the cycle.
• When the cell requires additional ornithine, it is synthesized from glucose via glutamate.

Arginine is a nonessential amino acid in adults. It is synthesized from glucose via ornithine and the first four reactions of the urea cycle.

Regulation of Urea cycle

• Carbamoyl phosphate synthetase I catalyzes the rate-limiting step of the cycle and is stimulated by N -acetylglutamate.
• Although the liver normally has a great capacity for urea synthesis, the enzymes of the urea cycle are induced if a high-protein diet is consumed for 4 days or more.

Purpose of the Urea cycle is it allows for the excretion of NH₄+ by transforming ammonia into urea, which is then excreted by the kidneys.

Enzymes involved in Urea cycle are

• Carbamoyl phosphate synthetase I: Converts ammonium and bicarbonate into carbamoyl phosphate. This is the rate-limiting step in the urea cycle. This reaction requires two ATP and occurs in the mitochondria.
• Ornithine transcarbamoylase: Combines ornithine and carbamoyl phosphate to form citrulline. Located in mitochondria.
• Argininosuccinate synthetase: Condenses citrulline with aspartate to form arginosuccinate. This reaction occurs in the cytosol and requires one ATP.
• Argininosuccinate lyase: Splits argininosuccinate into arginine and fumarate. Occurs in the cytosol.
• Arginase: Cleaves arginine into one molecule of urea and ornithine in the cytosol. The ornithine is then transported back into the mitochondria for entry back into the cycle.