Amino acid sequences transamination

Longenecker and Snell (274) have proposed a sequence of reactions shown in Fig. 1 to explain nonenzymic transamination systems involving pyridoxamine and pyridoxal. The same mechanism has been proposed by these authors to account for the above observations in the case of enzymic transamination. In the latter case, the apoenzyme is considered to play the role of the metal (M in Fig. 1). While there have been some reports suggesting the requirement for a metal in enzymic transamination systems (see 273) the evidence to date is inadequate on this point. Until transaminases of very high purity are prepared this will remain a moot point. A further feature of the reaction mechanism proposed by Longenecker and Snell is that an explanation is offered for the observations by Konikova, et al. (289) and Hilton et al. (290,291) on the labilization of the a-hydrogen atom of the substrate amino acid during transamination. 

A second mode of reaction of the quinonoid-carban-ionic intermediate is utilized by plants which synthesize an enzyme that acts on the amino acid S -adenosylmethionine to form a cyclic three-membered ring compound aminocy-clopropane carboxylic acid. This is a major plant hormone. In a third type of reaction a proton is added back to the coenzyme itself (see Fig. 14) to form what is called a ketimine (not illustrated). This is a Schiff base of pyridox-amine phosphate (PMP, Fig. 5) with an a-oxoacid and is an essential intermediate compound in the important process of transamination (Fig. 14). This process is utilized by all living organisms both in the synthesis of amino acids and in the breakdown of excesses of amino acids. The human body forms several amino acids via transamination. As shown in Fig. 15, this is a reversible sequence involving a cyclic interconversion of PLP and PMP in reaction steps of the type illustrated in Fig. 14. 

Many other amino acids are degraded in similar ways. In most cases the sequence is initiated by transamination to the corresponding 2-oxoacid. Beta oxidation and breakdown to such compounds as pyruvate and acetyl-CoA follows. 

There is an important biochemical counterpart of the deamination reaction that utilizes pyridoxal phosphate, 7, as the aldehyde. Each step in the sequence is catalyzed by a specific enzyme. The a-amino group of the amino acid combines with 7 and is converted to a keto acid. The resulting pyridoxamine then reacts to form an imine with a different a-keto acid, resulting in formation of a new a-amino acid and regenerating 7. The overall process is shown in Equation 25-6 and is called transamination. It is a key part of the process whereby amino acids are metabolized. 

A bacterial aminotransferase [46] promotes a decarboxylative transamination reaction with a-aminoisobutyrate in the presence of pyruvate. The reaction occurs via the sequence of Fig. 12 involving an initial cleavage of the Q-COjH bond in the substrate pyridoxal-P Schiff base complex (Fig. 12, 1) followed by reprotonation at C-4 of the coenzyme to give the pyridoxamine-P-enzyme complex (Fig. 12, 4) that participates in the transamination of pyruvate. However, the enzyme will also transform L-alanine at a significant rate by a half-transamination reaction into pyruvate, thereby implying that it is now the C -H bond of the amino acid that is first broken. 

Like homogeneous catalysis, the removal of a-hydrogen of the amino acid fragment by OH ions, the local concentration of which is apparently high in the polymer phase, is probably the rate-determining step of heterogeneous racemization. Under similar conditions, the rate of a-amino acid racemization decreases in the sequence Ala = Ser>Phe>Nva>Lys>Val, and correlates with the rate of substrate racemization in the presence of Schiff bases and transamination of amino acids by pyridoxal phosphate. 

A principal question, the monitoring of the effluent from the column remains to be discussed here. Small amounts of the individual amino acids emerging in the sequence of their elution have to be revealed and quantitated. In earlier procedures these amounts were in the range of 10 to 100 nanomoles but with improvements in the methodology much smaller samples can now be applied. The classical color reagent for the detection of nanomole quantities is ninhydrin, which reacts with amino acids in a transamination-decarboxylation reaction to yield Ruhemann s purple ... 

Adenosine deaminase converts adenosine monophosphate back to inosine monophosphate, liberating ammonia. This sequence of reactions thus provides a pathway for the deamination of a variety of amino acids, linked to transamination, similar to those shown in Figure 9.9 for transamination linked to glutamate dehydrogenase or glycine oxidase. 

In summary, the C skeleton of the amino acids is derived from carbohydrate metabolism. This means that whole families of amino acids can be traced back to one intermediate. The introduction of the amino group into the C skeleton can be considered to occur in a sequence of three main steps reduction of nitrate, reductive amination of a-ketoglutarate and transamination. 

Transamination Hydrolysis of the a-carbon-amino bond of the ketimine formed by deprotonation of the a-carbon of the amino acid results in the release of the 2-oxo-acid corresponding to the amino acid substrate and leaves pyridoxamine phosphate at the catalytic site of the enzyme. This is the half-reaction of transamination. The process is completed by reaction of pyridoxamine phosphate with a second oxo-acid substrate, forming an intermediate ketimine, followed by the reverse of the reaction sequence shown in Figure 3, releasing the amino acid corresponding to this second substrate after displacement from the aldimine by the reactive lysine residue to reform the internal Schiff base. 

The catabolism of lysine merges with that of tryptophan at the level of (3-ketoadipic acid. Both metabolic pathways are identical from this point on and lead to the formation of acetoacetyl-CoA (Figure 20.21). Lysine is thus ketogenic. It does not transaminate in the classic way. Lysine is a precursor of carnitine the initial reaction involves the methylation of e-amino groups of protein-bound lysine with SAM. The N-methylated lysine is then released proteolytically and the reaction sequence to carnitine completed. See Equation (19.6) for the structure of carnitine. 

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