Lecture 8_Biosynthesis of Nucleotides and Amino Acids_PDF

UNIVERSITY OF GUYANA FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BIO 3110 BIOCHEMISTRY II – Intermediary Metabolism LECTURE 8: Biosynthesis of Nucleotides and Amino Acids  Synthesis of Nucleotides Nucleotides...

UNIVERSITY OF GUYANA FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY BIO 3110 BIOCHEMISTRY II – Intermediary Metabolism LECTURE 8: Biosynthesis of Nucleotides and Amino Acids  Synthesis of Nucleotides Nucleotides are some of the largest monomers that have to be made by the cell. Their synthesis involves many steps and large amounts of energy - under tight regulatory control in the cell. Organisms need to make just the right amount of each base; if too much is made, energy is wasted, if too little, DNA replication and cellular metabolism come to a halt. NB: Nucleosides contain only sugar and a base whereas nucleotides contain sugar, base and a phosphate group as well.  Functions of Nucleotides Nucleotides serve various metabolic functions. For example, they are: Substrates (building blocks) for nucleic acid biosynthesis and repair, The main storage form of “high energy phosphate”, Components of many “so-called” co-enzymes (NAD, NADP, FAD, CoA), Components of many activated metabolic intermediates, Major allosteric effectors (such as AMP, ADP, ATP, GTP), Major second messengers Precursors for the biosynthesis of a variety of important compounds (such as histidine).  Formation of purine/pyrimidine nucleotides There are two pathways for the biosynthesis of nucleotides: 1. De-novo synthesis: biochemical pathway which nucleotides are synthesised from simple precursor molecules. 2. Salvage pathway: used to recover bases and nucleotides formed during the degradation of RNA and DNA.  Synthesis of Nucleotides All nucleotides contain a ribose sugar and phosphate that form the backbone of DNA and RNA. These are synthesized from ribose 5-phosphate, a central metabolite of the pentose phosphate pathway. In this single-step reaction, two of the phosphates of ATP are transferred to ribose 5-phosphate to form 5-phosphoribosyl-1-pyrophosphate (PRPP). This intermediate is required for the biosynthesis of purines, pyrimidines, NAD, histidine, and tryptophan. It plays a critical role in anabolism. De novo synthesis of purine nucleotides Ribose 5-phosphate from the pentose phosphate pathway is used as the first substrate in the synthesis of purines. Synthesis begins with assembling a purine ring on ribose 5- phosphate, i.e. purine is being formed in the progress of synthesis. ATP and GTP are required as sources of energy.  Synthesis of Purines Purine synthesis starts with the addition of ammonia from glutamine to PRPP → 5 – phosphoribosyl-1-amine. In six more steps the five-membered ring of purines is synthesized with a net cost of five ATP. Glycine, tetrahydrofolate (a one carbon carrier that donates a methyl group, coenzyme associated with Vitamin B9/folic acid), glutamine, and bicarbonate (HCO3-) all donate parts of the five membered ring, the final product being 5'- phosphoribosyl-5- aminoimidazole-4-carbonate (CAIR).  5 – phosphoribosyl-1-amine Regulatory enzyme: Glutamine phosphoribosyl pyrophosphate (PRPP) amidotransferase  Synthesis of Purines In the next series of reactions, the 6 membered ring of purine is added to CAIR creating Inosine 5'-monophosphate (IMP)/inosinate. This costs an additional ATP with the members of the ring coming from aspartate and tetrahydrofolate. IMP is a purine - compare it to adenosine or guanine and you will see they are very similar.  Both rings are now closed Aspartate Tetrahydrofolate (donates 1C)  Synthesis or guanine or adenine involves the addition of an amino group in the appropriate place. To form guanine monophosphate (GMP), IMP is first oxidized to xanthosine monophosphate. This adds a ketone group (see figure) that is attacked by the ammonia on glutamine in the next reaction yielding GMP. The cost to the cell is one ATP. In the synthesis of adenosine monophosphate (AMP), IMP first combines with aspartate and in a second reaction the combination is split into fumarate and AMP. The cost to the cell is one GTP. It is interesting to note that synthesis of ATP from IMP requires GTP as an energy source and not ATP. Note the use of GTP instead of ATP to catalyze the first step in the reaction. Ketone formed, will be attacked by ammonia from glutamine Note the extra energy that is required in the form of pyrophosphate to drive the reaction. Synthesis of Pyrimidines  Synthesis of Pyrimidines Pyrimidine synthesis is simpler due to the single ring of these nucleotides. The synthesis of pyrimidines begins by combining glutamine, 2 ATP and bicarbonate to form glutamate, 2 ADP and carbamoyl phosphate (this does not come from the urea cycle but is synthesized separately in the cytosol of all tissues). Aspartate next reacts with carbamoyl phosphate forming carbamoyl aspartate. In the critical third step, carbamoyl aspartate is cyclized to form the recognizable 6-membered ring of pyrimidines.  Synthesis of Pyrimidines Later in the pathway, PRPP is combined with the six-membered ring to form orotidine 5'-phosphate. Removal of CO2 results in the formation of Uridine monophosphate (UMP). Two kinase reactions (addition of phosphate) finally form uridine triphosphate (UTP). An addition of an amino group to UTP results in the formation of cytidine triphosphate (CTP). The synthesis of UTP uses 4 ATP (not counting the formation of PRPP) and making CTP adds an extra ATP to the cost.  Double bond is formed! Vital for pyrimidine formation! Ring is then attached to the ribose-5- phosphate Ring is formed!  Uridine triphosphate Uridine monophosphate Cytidine triphosphate  Synthesis of Thymine Nucleotides Uridine nucleotides are also the precursors for de novo synthesis of the thymine nucleotides. UDP → deoxyuridine monophosphate (dUDP) is catalysed by ribonucleotide reductase. Subsequently, dUDP is converted to dUMP. The methylation of dUMP to generate deoxythymidine monophosphate (dTMP) is catalysed by thymidylate synthase using N5, N10-methylene THF as a methyl donor. Regulation of Nucleotide Synthesis Disorders of Nucleotide Metabolism  Biosynthesis of Amino Acids ▪ An amino acid is organic molecule that contains a carboxyl group, –COOH ,an amine group –NH2, as well as R-group. ▪ Amino acids derived from proteins have the amino group on the alpha (a) carbon i.e; the carbon atom next to the carboxyl group. ▪ The amino acids differ in the nature of R group attached to a carbon atom. The nature of R-group determines the properties of proteins. ▪ The major key elements of amino acids are carbon, hydrogen, nitrogen, oxygen.  Biosynthesis of Amino Acids Amino acids are the structural units that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These polymers are linear and unbranched, with each amino acid within the chain attached to two neighbouring amino acids. In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation.  Types of amino acids based on nutritional requirements E s s e n t i a l amino acids: Essential amino acids are unable to be synthesised by the body. They must obtained from food and their sources are plants and microbes. Non-essential amino acids: If they are not supplied in our diet we can synthesise them. Most of the amine group comes from glutamate and glutamine. Some of them are synthesised from essential amino acids.  Amino acids precursors are intermediates in glycolysis, the citric acid cycle, or the pentose phosphate pathway. They are used to make nucleotides. Histidine comes from Ribose 5-phosphate which is a product of the pentose phosphate pathway. Serine comes from 3-phosphoglycerate and from serine we can make more amino acids like glycine and cysteine. Tryptophan and tyrosine come from phosphoenolpyruvate. Pyruvate used to make alanine, valine, leucine and isoleucine. Some amino acids are both glucogenic and ketogenic, and are known as amphibolic. These include: phenylalanine, isoleucine, threonine, tryptophan, and tyrosine.  We can make aspartate from oxaloacetate and we can use aspartate to make other amino acids like aspargine, methionine, threonine and lysine. Glutamate is synthesized from α- ketoglutarate. And from glutamate, we can make more amino acids like glutamine, proline and arginine. Synthesis of Non- essential amino acids  Glutamate Synthesis from α-ketoglutarate Glutamate is synthesized by the reductive amination of α- ketoglutarate catalyzed by glutamate dehydrogenase (GDH); it is thus a nitrogen-incorporating reaction. When energy and carbon levels are high, glutamate can incorporate nitrogen (from NH +) into α-ketoglutarate uising NADPH generated from the Pentose Phosphate Pathway. When energy levels are reduced, glutamate can be oxidatively deaminated in the opposite direction allowing α - k e t o g l u t a r a t e to be utilized in the TCA cycle for the production of energy.  Glutamine Synthesis From Glutamate Glutamine is synthesized from glutamate via the action of glutamine synthetase. The synthesis of glutamine is a two-step reaction. Glutamate is first "activated" to a gema- glutamylphosphate intermediate, followed by a reaction in which NH 3 displaces the phosphate group.  Aspartate Synthesis From Oxaloacetate Aspartate is synthesized by the transfer of an ammonia group from glutamate to oxaloacetate. Aspartate can be formed in a transamination reaction. The transamination reaction is catalyzed by aspartate transaminase, AST. This reaction uses the α-keto acid oxaloacetate as the amino acceptor and glutamate as the primary amino group donor.  Synthesis of Asparagine from Aspartate Asparagine is synthesized from aspartate via an amidotransferase reaction catalyzed by asparagine synthetase.  Alanine s y n t h e s i s from P y r u v a t e There are two main pathways to the production of muscle alanine: directly from protein degradation, and via the transamination of pyruvate by alanine transaminase (ALT).  Synthesis of serine Serine can be derived from the glycolytic intermediate, 3-phosphoglycerate, in a three-step reaction pathway. The first reaction is catalyzed by phosphoglycerate dehydrogenase (PHGDH) in which oxidation of the hydroxyl group of 3- phosphoglycerate by NAD+ produce 3- phosphohydroxy pyruvate. The second reaction is a simple transamination catalyzed by phosphoserine aminotransferase (PSAT) which utilizes glutamate as the amino donor and releases α- ketoglutarate. The last step in the reaction pathway is catalyzed by phosphoserine phosphatase (PSPH). Finally, hydrolysis and removal of the phosphate group yields serine.  Synthesis of Glycine from serine The main pathway to glycine is a one-step reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT). This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N5,N10-methylene-THF.  Synthesis of Essential Amino Acids The synthetic pathways for the essential amino acids are: (1) present only in microorganisms (2) considerably more complex than non-essential amino acids (3) Use familiar metabolic precursors (4) Show species variation For purposes of classification, consider the following 4 "families" which are based upon common precursors: (1) Aspartate Family: lysine, methionine, threonine (2) Pyruvate Family: leucine, isoleucine, valine (3) Aromatic Family: phenylalanine, Tyrosine, Tryptophan (4) Histidine  Synthesis of valine and leucine In the 1st step, acetolactate synthase transfers the acyl group of pyruvate to another molecule of pyruvate, forming acetolactate. In the 2nd step acetolactate is converted into 2,3-di hydroxyisovalerate in the presence of acetohydroxyacid reductoisomerase. In 3rd step, dihydroxy acid dehydratase enzyme converts 2,3-di hydroxyisovalerate into 2-keto-isovalerate. In 4th step 2-keto-isovalerate is converted into Valine and leucine with the help of transferase enzymes.  S y n t h e s i s o f phenylalanine and t y r o s i n e An alternative pathway of phenylalanine and tyrosine biosynthesis from chorismate (an important biochemical intermediate in plants and microorganisms) has been characterized in bacteria, fungi and higher plants. The alternative pathway called pretyrosine (or arogenate) pathway involves the transamination of prephenate to form pretyrosine (arogenate). Tyrosine and phenylalanine can then be formed from prephenate by prephenate dehydrogenase and prephenate dehydratase, respectively. 2. Dehydrogenases are a group of biological catalysts (enzymes) that mediate in biochemical reactions removing hydrogen atoms [H] instead of oxygen [O] 3. Dehydratases are a group of lyase enzymes that form double and triple bonds in a substrate through the removal of water END OF LECTURE 8