The following points highlight five reactions on direct assimilation of ammonia into organic compounds. The reactions are: 1. Reductive Amination 2. Transamination 3. Formation of Amides 4. Direct Amination of Fumarate 5. Synthesis of Carbamoyl Phosphate.
Reaction # 1. Reductive Amination:
The general principle of this process is the formation of amino acid from a keto acid reacting with ammonia as follows:
Therefore, the amino acids are synthesized in two steps—the union of ammonia with a keto acid to form an imino acid which is subsequently reduced to an amino acid.
The former step proceeds spontaneously, but the latter reaction is catalyzed by the dehydrogenase enzyme that requires the presence of reduced NAD+. Glutamic acid is readily formed from α-ketoglutaric acid by the same mechanism catalyzed by the enzyme glutamate dehydrogenase with reduced coenzyme (NADH) (Fig. 10.14).
This reaction of reductive amination is of central importance because of the high proportion of glutamate is formed in this manner. It is the major part of entry of ammonia into the metabolism.
Sims et al. (1968) have indicated that 75-80 per cent of the nitrogen assimilated follows this pathway. Aspartic acid and alanine are the other amino acids which may arise from oxaloacetic acid and pyruvic acid in a similar way (Fig. 10.15 and Fig. 10.16).
Amino acid synthesis through reductive amination is intimately related to the process of aerobic respiration and organic acid metabolism. During aerobic respiration the conversions of α-ketoglutaric acid to glutamic acid, oxaloacetic acid to aspartic acid and pyruvic acid to alanine are considered as side reactions of Krebs cycle.
Amino acid synthesis may also take place when a keto acid reacts with hydroxylamine. An intermediary product, called oxime of that particular keto acid, is formed.
This type of reaction is found only in symbiotic association and not in other plants.
Reaction # 2. Transamination:
There is another major route for the entry of an amino group into the keto acid, known as transamination. It involves the transfer of a-amino group from one amino acid to a carboxyl group of a keto acid without the intermediate formation of free ammonia. Glutamic acid is a major amino acid.
As soon as the inorganic nitrogen enters through the amination system with a-keto glutaric acid to form glutamic acid, it becomes available for the synthesis of other plant amino acids by transamination mechanism. The enzyme responsible for such reaction is termed as amino-transferase or transaminase mentioning the name of the specific substrates on which it reacts.
The general plan of the reaction is as follows:
Transaminase enzymes of varying degrees of specificity are found to catalyse the different transamination reactions. The important transaminases are aspartate transaminase, alanine transaminase, leucine transaminase and tyrosine transaminase.
Reaction # 3. Formation of Amides:
Amides are formed from amino acids by replacement of the hydroxyl part of the amino acid by a NH2 radical. The amidation reactions are catalysed by the specific enzymes.
The most important plant amides are glutamine and asparagine’s, which are produced by the amination of glutamic acid and aspartic acid respectively. Sims et al., (1968), have estimated that 10-12 per cent of the nitrogen assimilated through glutamate is incorporated into glutamine (Fig. 10.17).
The reaction is catalysed by the enzyme glutamine synthetase in the presence of Mg-ATP. The reaction takes place in two steps:
(i) Formation of γ-Glutamyl Phosphate:
(ii) Formation of Glutamine from γ-Glutamyl Phosphate:
Asparagine, on the other hand, is derived from aspartic acid:
However, asparagine synthetase has not yet been successfully obtained in purified form. Asparagine synthesis also follows another route in which the amide amino group of glutamine is transferred to the β-carboxyl group of aspartic acid.
Reaction # 4. Direct Amination of Fumarate:
It has been reported that an enzyme called aspartase is present in some plants. This enzyme helps in the formation of aspartic acid by the amination of fumaric acid.
Reaction # 5. Synthesis of Carbamoyl Phosphate:
The synthesis of carbamoyl phosphate is another major route for the assimilation of free ammonia into organic compounds. The reaction is very complex and is catalyzed by the enzyme carbamoyl kinase which is present in the cytosol of bacteria and fungi.
The carbamoyl phosphate is a very unstable compound and it is an intermediate in the reaction of NH3 and CO2 with ornithine to form citrulline. The enzyme carbamoyl kinase has been shown to catalyze the phosphorylation of the ammonium salt of carbamic acid. The reaction is relatively endergonic.
In the mammalian system, another enzyme carbamoyl phosphate synthetase present in the mitochondrial matrix catalyzes the formation of carbamoyl phosphate from NH3 and CO2. In this reaction two molecules of ATP are required to form each molecule of carbamoyl phosphate. The reaction is essentially irreversible.
2ATP + CO2 + NH3 + H2O → Carbamoyl phosphate + 2ADP + Pi
Carbamoyl phosphate made by either of these processes serves as a donor of the carbamoyl group in citrulline biosynthesis and in pyrimidine biosynthesis.