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.

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 Bicarbonate Buffer System

This is the main extracellular buffer system which (also) provides a means for the necessary removal of the CO2 produced by tissue metabolism. The bicarbonate buffer system is the main buffer in blood plasma and consists of carbonic acid as proton donor and bicarbonate as proton acceptor :

 H₂CO₃ = H⁺ + HCO₃^–

If there is a change in the ratio in favour of H₂CO₃, acidosis results.

This change can result from a decrease in [HCO₃ ⁻ ] or from an increase in [H₂CO₃ ]

Most common forms of acidosis are metabolic or respiratory

Metabolic acidosis is caused by a decrease in [HCO₃ ⁻ ] and occurs, for example, in uncontrolled diabetes with ketosis or as a result of starvation.

Respiratory acidosis is brought about when there is an obstruction to respiration (emphysema, asthma or pneumonia) or depression of respiration (toxic doses of morphine or other respiratory depressants)

Alkalosis results when [HCO₃ ⁻ ] becomes favoured in the bicarbonate/carbonic acid ratio

Metabolic alkalosis occurs when the HCO₃ ⁻  fraction increases with little or no concomitant change in H₂CO₃

Severe vomiting (loss of H+ as HCl) or ingestion of excessive amounts of sodium bicarbonate (bicarbonate of soda) can produce this condition

Respiratory alkalosis is induced by hyperventilation because an excessive removal of CO2 from the blood results in a decrease in [H₂CO₃ ]

Alkalosis can produce convulsive seizures in children and tetany, hysteria, prolonged hot baths or lack of O2 as high altitudes.

The pH of blood is maintained at 7.4 when the buffer ratio [HCO3 − ] / [ H₂CO₃] becomes 20

Glycolysis Pathway

The reactions of Glycolysis take place in the cytosol of cells.

Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially, there is energy input corresponding to cleavage of two ~P bonds of ATP. 

1. Hexokinase catalyzes:  glucose + ATP → glucose-6-phosphate + ADP

ATP binds to the enzyme as a complex with Mg⁺⁺.

The reaction catalyzed by Hexokinase is highly spontaneous 

2. Phosphoglucose Isomerase catalyzes: 

glucose-6-phosphate (aldose) → fructose-6-phosphate (ketose)

The Phosphoglucose Isomerase mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure .

3. Phosphofructokinase catalyzes: 

fructose-6-phosphate + ATP  → fructose-1,6-bisphosphate + ADP

The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated. 

4. Aldolase catalyzes: 

fructose-1,6-bisphosphate   → dihydroxyacetone phosphate + glyceraldehyde-3-phosphate

The Aldolase reaction is an aldol cleavage, the reverse of an aldol condensation.

5. Triose Phosphate Isomerase (TIM) catalyzes

dihydroxyacetone phosphate (ketose) → glyceraldehyde-3-phosphate (aldose)

Glycolysis continues from glyceraldehydes-3-phosphate

The equilibrium constant (K_eq) for the TIM reaction favors dihydroxyacetone phosphate, but removal of glyceraldehyde-3-phosphate by a subsequent spontaneous reaction allows throughput. 

6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:

glyceraldehyde-3-phosphate + NAD⁺ + P_i  → 1,3,bisphosphoglycerate + NADH + H⁺

This is the only step in Glycolysis in which NAD⁺ is reduced to NADH

A cysteine thiol at the active site of Glyceraldehyde-3-phosphate Dehydrogenase has a role in catalysis . 

7. Phosphoglycerate Kinase catalyzes:

1,3-bisphosphoglycerate + ADP  →  3-phosphoglycerate + ATP

This transfer of phosphate to ADP, from the carboxyl group on 1,3-bisphosphoglycerate, is reversible

8. Phosphoglycerate Mutase catalyzes:  3-phosphoglycerate → 2-phosphoglycerate

Phosphate is shifted from the hydroxyl on C3 of 3-phosphoglycerate to the hydroxyl on C2.  

9. Enolase catalyzes:  2-phosphoglycerate  → phosphoenolpyruvate + H₂O

This Mg⁺⁺-dependent dehydration reaction is inhibited by fluoride. Fluorophosphate forms a complex with Mg⁺⁺ at the active site .

10. Pyruvate Kinase catalyzes:  phosphoenolpyruvate + ADP  → pyruvate + ATP

This transfer of phosphate from PEP to ADP is spontaneous. 

Balance sheet for high energy bonds of ATP: 

• 2 ATP expended
• 4 ATP produced (2 from each of two 3C fragments from glucose) 
• Net Production of 2~ P bonds of ATP per glucose

Acyl-CoA Synthases (Thiokinases), associated with endoplasmic reticulum membranes and the outer mitochondrial membrane, catalyze activation of long chain fatty acids, esterifying them to coenzyme A, as shown at right. This process is ATP-dependent, and occurs in 2 steps. There are different Acyl-CoA Synthases for fatty acids of different chain lengths. 

Exergonic hydrolysis of PP_i (P~P), catalyzed by Pyrophosphatase, makes the coupled reaction spontaneous. Overall, two ~P bonds of ATP are cleaved during fatty acid activation. The acyl-coenzyme A product includes one "high energy" thioester linkage.

Summary of fatty acid activation:

• fatty acid + ATP → acyl-adenylate + PP_i
  PP_i  → P_i
• acyladenylate + HS-CoA → acyl-CoA + AMP

Overall: fatty acid + ATP + HS-CoA → acyl-CoA + AMP +  2 P_i

For most steps of the b-Oxidation Pathway, there are multiple enzymes specific for particular fatty acid chain lengths.

Fatty acid b-oxidation is considered to occur in the mitochondrial matrix. Fatty acids must enter the matrix to be oxidized. However enzymes of the pathway specific for very long chain fatty acids are associated with the inner mitochondrial membrane (facing the matrix).

Fatty acyl-CoA formed outside the mitochondria can pass through the outer mitochondrial membrane, which contains large VDAC channels, but cannot penetrate the mitochondrial inner membrane.

Transfer of the fatty acid moiety across the inner mitochondrial membrane involves carnitine.

Carnitine Palmitoyl Transferases catalyze transfer of a fatty acid between the thiol of Coenzyme A and the hydroxyl on carnitine.

Carnitine-mediated transfer of the fatty acyl moiety into the mitochondrial matrix is a 3-step process, as presented below.

1. Carnitine Palmitoyl Transferase I, an enzyme associated with the cytosolic surface of the outer mitochondrial membrane, catalyzes transfer of a fatty acid from ester linkage with the thiol of coenzyme A to the hydroxyl on carnitine.
2. Carnitine Acyltransferase, an antiporter in the inner mitochondrial membrane, mediates transmembrane exchange of fatty acyl-carnitine for carnitine.
3. Within the mitochondrial matrix (or associated with the matrix surface of the inner mitochondrial membrane, Carnitine Palmitoyl Transferase II catalyzes transfer of the fatty acid from carnitine to coenzyme A. (Carnitine exits the matrix in step 2.) The fatty acid is now esterified to coenzyme A within the mitochondrial matrix

Control of fatty acid oxidation is exerted mainly at the step of fatty acid entry into mitochondria.

Malonyl-CoA inhibits Carnitine Palmitoyl Transferase I. (Malonyl-CoA is also a precursor for fatty acid synthesis). Malonyl-CoA is produced from acetyl-CoA by the enzyme Acetyl-CoA Carboxylase

AMP-Activated Kinase, a sensor of cellular energy levels, catalyzes phosphorylation of Acetyl-CoA Carboxylase under conditions of high AMP (when ATP is low). Phosphorylation inhibits Acetyl-CoA Carboxylase, thereby decreasing malonyl-CoA production.

The decrease in malonyl-CoA concentration releases Carnitine Palmitoyl Transferase I from inhibition. The resulting increase in fatty acid oxidation generates acetyl-CoA for entry into Krebs cycle, with associated production of ATP

Vitamin B12: Cobalamin

Vitamin B12, also known as cobalamin, aids in the building of genetic material, production of normal red blood cells, and maintenance of the nervous system.

RDA The Recommended Dietary Allowance (RDA) for vitamin B12 is 2.4 mcg/day for adult males and females

Vitamin B12 Deficiency

Vitamin B12 deficiency most commonly affects strict vegetarians (those who eat no animal products), infants of vegan mothers, and the elderly. Symptoms of deficiency include anemia, fatigue, neurological disorders, and degeneration of nerves resulting in numbness and tingling.