Amino Acid Biosynthesis

Glutamate and Aspartate

Glutamate and aspartate are synthesized from their widely distributed a-keto acid precursors by simple 1-step transamination reactions. The former catalyzed by glutamate dehydrogenase and the latter by aspartate aminotransferase, AST. Aspartate is also derived from asparagine through the action of asparaginase. The importance of glutamate as a common intracellular amino donor for transamination reactions and of aspartate as a precursor of ornithine for the urea cycle is described in the Nitrogen Metabolism page.
 

Alanine and the Glucose-Alanine Cycle

Role in protein synthesis,

Alanine is second only to glutamine in prominence as a circulating amino acid.. When alanine transfer from muscle to liver is coupled with glucose transport from liver back to muscle, the process is known as the glucose-alanine cycle. The key feature of the cycle is that in 1 molecule, alanine, peripheral tissue exports pyruvate and ammonia (which are potentially rate-limiting for metabolism) to the liver, where the carbon skeleton is recycled and most nitrogen eliminated.

There are 2 main pathways to production of muscle alanine: directly from protein degradation, and via the transamination of pyruvate by alanine transaminase, ALT (also referred to as serum glutamate-pyruvate transaminase, SGPT).

glutamate + pyruvate <-------> a-KG + alanine

Cysteine Biosynthesis

The sulfur for cysteine synthesis comes from the essential amino acid methionine. A condensation of ATP and methionine catalyzed by methionine adenosyltransferase yields S-adenosylmethionine

Tyrosine Biosynthesis

Tyrosine is produced in cells by hydroxylating the essential amino acid phenylalanine. This relationship is much like that between cysteine and methionine. Half of the phenylalanine required goes into the production of tyrosine; if the diet is rich in tyrosine itself, the requirements for phenylalanine are reduced by about 50%.

Phenylalanine hydroxylase is a mixed-function oxygenase: one atom of oxygen is incorporated into water and the other into the hydroxyl of tyrosine. The reductant is the tetrahydrofolate-related cofactor tetrahydrobiopterin, which is maintained in the reduced state by the NADH-dependent enzyme dihydropteridine reductase (DHPR).

Ornithine and Proline Biosynthesis

Glutamate is the precursor of both proline and ornithine, with glutamate semialdehyde being a branch point intermediate leading to one or the other of these 2 products. While ornithine is not one of the 20 amino acids used in protein synthesis, it plays a significant role as the acceptor of carbamoyl phosphate in the urea cycle

Serine Biosynthesis

The main pathway to serine starts with the glycolytic intermediate 3-phosphoglycerate. An NADH-linked dehydrogenase converts 3-phosphoglycerate into a keto acid, 3-phosphopyruvate, suitable for subsequent transamination. Aminotransferase activity with glutamate as a donor produces 3-phosphoserine, which is converted to serine by phosphoserine phosphatase.
 

Glycine Biosynthesis

The main pathway to glycine is a 1-step reaction catalyzed by serine hydroxymethyltransferase. This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N⁵,N¹⁰-methylene-THF. Glycine produced from serine or from the diet can also be oxidized by glycine cleavage complex, GCC, to yield a second equivalent of N⁵,N¹⁰-methylene-tetrahydrofolate as well as ammonia and CO₂.

Glycine is involved in many anabolic reactions other than protein synthesis including the synthesis of purine nucleotides, heme, glutathione, creatine and serine.

Aspartate/Asparagine and Glutamate/Glutamine Biosynthesis

Glutamate is synthesized by the reductive amination of a-ketoglutarate catalyzed by glutamate dehydrogenase; it is thus a nitrogen-fixing reaction. In addition, glutamate arises by aminotransferase reactions, with the amino nitrogen being donated by a number of different amino acids. Thus, glutamate is a general collector of amino nitrogen.

Aspartate is formed in a transamintion reaction catalyzed by aspartate transaminase, AST. This reaction uses the aspartate a-keto acid analog, oxaloacetate, and glutamate as the amino donor. Aspartate can also be formed by deamination of asparagine catalyzed by asparaginase.

Asparagine synthetase and glutamine synthetase, catalyze the production of asparagine and glutamine from their respective a-amino acids. Glutamine is produced from glutamate by the direct incorporation of ammonia; and this can be considered another nitrogen fixing reaction. Asparagine, however, is formed by an amidotransferase reaction.

Aminotransferase reactions are readily reversible. The direction of any individual transamination depends principally on the concentration ratio of reactants and products. By contrast, transamidation reactions, which are dependent on ATP, are considered irreversible. As a consequence, the degradation of asparagine and glutamine take place by a hydrolytic pathway rather than by a reversal of the pathway by which they were formed. As indicated above, asparagine can be degraded to aspartate

The basic characteristics of enzymes includes

(i) Almost all the enzymes are proteins and they follow the physical and chemical reactions of proteins (ii) Enzymes are sensitive and labile to heat

(iii) Enzymes are water soluble

(iv) Enzymes could be precipitated by protein precipitating agents such as ammonium sulfate and trichloroacetic acid.

Gluconeogenesis

It is the process by which Glucose or glycogen is formed from non carbohydrate substances.

Gluconeogenesis occurs mainly in liver.

Gluconeogenesis inputs:  
The source of pyruvate and oxaloacetate for gluconeogenesis during fasting or carbohydrate starvation is mainly amino acid catabolism. Some amino acids are catabolized to pyruvate, oxaloacetate, Muscle proteins may break down to supply amino acids. These are transported to liver where they are deaminated and converted to gluconeogenesis inputs. 
Glycerol, derived from hydrolysis of triacylglycerols in fat cells, is also a significant input to gluconeogenesis

Glycolysis & Gluconeogenesis pathways are both spontaneous If both pathways were simultaneously active within a cell it would constitute a "futile cycle" that would waste energy

Glycolysis yields 2~P bonds of ATP.
Gluconeogenesis expends 6~P  bonds of ATP and GTP.
A futile cycle consisting of both pathways would waste 4 P.bonds per cycle.To prevent this waste, Glycolysis and Gluconeogenesis pathways are reciprocally regulated.

Step 1.  Acyl-CoA Dehydrogenase catalyzes oxidation of the fatty acid moiety of acyl-CoA, to produce a double bond between carbon atoms 2 and 3.

There are different Acyl-CoA Dehydrogenases for short (4-6 C), medium (6-10 C), long and very long (12-18 C) chain fatty acids. Very Long Chain Acyl-CoA Dehydrogenase is bound to the inner mitochondrial membrane. The others are soluble enzymes located in the mitochondrial matrix.

FAD is the prosthetic group that functions as electron acceptor for Acyl-CoA Dehydrogenase. 

A glutamate side-chain carboxyl extracts a proton from the a-carbon of the substrate, facilitating transfer of 2 e^- with H⁺ (a hydride) from the b position to FAD. The reduced FAD accepts a second H⁺, yielding FADH₂

The carbonyl oxygen of the thioester substrate is hydrogen bonded to the 2'-OH of the ribityl moiety of FAD, giving this part of FAD a role in positioning the substrate and increasing acidity of the substrate a-proton

The reactive glutamate and FAD are on opposite sides of the substrate at the active site. Thus the reaction is stereospecific, yielding a trans double bond in enoyl-CoA.

FADH₂ of Acyl CoA Dehydrogenase is reoxidized by transfer of 2 electrons to an Electron Transfer Flavoprotein (ETF), which in turn passes the electrons to coenzyme Q of the respiratory chain.

Step 2. Enoyl-CoA Hydratase catalyzes stereospecific hydration of the trans double bond produced in the 1st step of the pathway, yielding L-hydroxyacyl-Coenzyme A

Step 3. Hydroxyacyl-CoA Dehydrogenase catalyzes oxidation of the  hydroxyl in the b position (C3) to a ketone. NAD⁺ is the electron acceptor.

Step 4. b-Ketothiolase (b-Ketoacyl-CoA Thiolase) catalyzes thiolytic cleavage.

A cysteine S attacks the b-keto C. Acetyl-CoA is released, leaving the fatty acyl moiety in thioester linkage to the cysteine thiol. The thiol of HSCoA displaces the cysteine thiol, yielding fatty acyl-CoA (2 C shorter).

A membrane-bound trifunctional protein complex with two subunit types expresses the enzyme activities for steps 2-4 of the b-oxidation pathway for long chain fatty acids. Equivalent enzymes for shorter chain fatty acids are soluble proteins of the mitochondrial matrix.

Summary of one round of the b-oxidation pathway:

fatty acyl-CoA + FAD + NAD⁺ + HS-CoA → 
            fatty acyl-CoA (2 C shorter) + FADH₂ + NADH + H⁺ + acetyl-CoA

The b-oxidation pathway is cyclic. The product, 2 carbons shorter, is the input to another round of the pathway. If, as is usually the case, the fatty acid contains an even number of C atoms, in the final reaction cycle butyryl-CoA is converted to 2 copies of acetyl-CoA

ATP production:

• FADH₂ of Acyl CoA Dehydrogenase is reoxidized by transfer of 2 e^- via ETF to coenzyme Q of the respiratory chain. H⁺ ejection from the mitochondrial matrix that accompanies transfer of 2 e^- from CoQ to oxygen, leads via chemiosmotic coupling to production of approximately 1.5 ATP. (Approx. 4 H⁺ enter the mitochondrial matrix per ATP synthesized.)
• NADH is reoxidized by transfer of 2 e^- to the respiratory chain complex I. Transfer of 2 e^- from complex I to oxygen yields approximately 2.5 ATP.
• Acetyl-CoA can enter Krebs cycle, where the acetate is oxidized to CO₂, yielding additional NADH, FADH₂, and ATP. 
• Fatty acid oxidation is a major source of cellular ATP

b-Oxidation of very long chain fatty acids also occurs within peroxisomes

FAD is electron acceptor for peroxisomal Acyl-CoA Oxidase, which catalyzes the first oxidative step of the pathway. The resulting FADH₂ is reoxidized in the peroxisome producing hydrogen peroxide FADH₂ + O₂ à FAD + H₂O₂

The peroxisomal enzyme Catalase degrades H₂O₂ by the reaction:
2 H₂O₂ → 2 H₂O + O₂
These reactions produce no ATP

Once fatty acids are reduced in length within the peroxisomes they may shift to the mitochondria to be catabolized all the way to CO₂. Carnitine is also involved in transfer of fatty acids into and out of peroxisomes

Clinical significance

Primary hyperparathyroidism is due to autonomous, abnormal hypersecretion of PTH in the parathyroid gland

Secondary hyperparathyroidism is an appropriately high PTH level seen as a physiological response to hypocalcemia.

A low level of PTH in the blood is known as hypoparathyroidism and is most commonly due to damage to or removal of parathyroid glands during thyroid surgery.

Pentose Phosphate Pathway (Hexose Monophosphate Shunt)

The pentose phosphate pathway is primarily an anabolic pathway that utilizes the 6 carbons of glucose to generate 5 carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO₂ and water. The primary functions of this pathway are:

• To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells.
• To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.
• Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates

Enzymes that function primarily in the reductive direction utilize the NADP⁺/NADPH cofactor pair as co-factors as opposed to oxidative enzymes that utilize the NAD⁺/NADH cofactor pair. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione. The conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source, therefore, any rapidly proliferating cell needs large quantities of NADPH.

Regulation: Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP⁺. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP⁺ stimulates the Pentose Phosphate Pathway, to replenish NADPH