Amino acid metabolism, enzymes

It is obvious that AOA can be considered the aminooxy analogue of glycine, while AOPP is the aminooxy analogue of phenylalanine. Both of these compounds can be expected to interfer with amino acid metabolizing enzymes carrying a carbonyl group(e.g. that of pyridoxal phosphate in the case of transaminases or dehydroalanine in... 

A subclass of lyases, involved in amino acid metabolism, utilizes pyridoxal 5-phosphate (PLP, 3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]-4-pyridinecarbaldehyde) as a cofactor for imine/ enamine-type activation. These enzymes are not only an alternative to standard fermentation technology, but also offer a potential entry to nonnatural amino acids. Serine hydroxymethyl-tansferase (SHMT EC 2.1.2.1.) combines glycine as the donor with (tetrahydrofolate activated) formaldehyde to L-serine in an economic yield40, but will also accept a range of other aldehydes to provide /i-hydroxy-a-amino acids with a high degree of both absolute and relative stereochemical control in favor of the L-erythro isomers41. 

Pyridoxal phosphate mainly serves as coenzyme in the amino acid metabolism and is covalently bound to its enzyme via a Schiff base. In the enzymatic reaction, the amino group of the substrate and the aldehyde group of PLP form a Schiff base, too. The subsequent reactions can take place at the a-, (3-, or y-carbon of the respective substrate. Common types of reactions are decarboxylations (formation of biogenic amines), transaminations (transfer of the amino nitrogen of one amino acid to the keto analog of another amino acid), and eliminations. 

Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially in transamination and decarboxylation. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. In addition, vitamin Bg is important in steroid hormone action where it removes the hormone-receptor complex from DNA binding, terminating the action of the hormones. In vitamin Bg deficiency, this results in increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D. 

Pantothenic acid is present in coenzyme A and acyl carrier protein, which act as carriers for acyl groups in metabolic reactions. Pyridoxine, as pyridoxal phosphate, is the coenzyme for several enzymes of amino acid metabolism, including the aminotransferases, and of glycogen phosphorylase. Biotin is the coenzyme for several carboxylase enzymes. 

A number of factors complicate the aerobic metabolism of amino acids—different enzymes may be used even for the same amino acid the enzymes may be inducible or constitutive depending on their function a-ketoacids may be produced by deamination or amines by decarboxylation. 

The high toxicity of AOA is due to its very high efficiency as a transaminase inhibitor (K =0.45 pM) as compared to its efficacy as a PAL inhibitor (K. = 120 pM) (48), making it impossible to effectively inhibit PAL iti vivo without also greatly inhibiting amino acid metabolism. Other pyridoxyl phosphate-requiring enzymes, such as ACC synthase (an enzyme involved in ethylene production) (49), are also more sensitive to AOA than to AOPP. 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

Other leukodystrophies are associated with the lysosomal and peroxisomal disorders in which specific lipids or other substances accumulate due to a deficiency in a catabolic enzyme - for example Krabbe s disease, meta-chromatic leukodystrophy (MLD) and adrenoleuko-dystrophy (ALD) [1,2]. (These are discussed in detail in Ch. 40.) Similarly, disorders of amino acid metabolism can lead to hypomyelination - for example phenylketonuria and Canavan s disease (spongy degeneration) [1, 2, 25] (Ch. 40). The composition of myelin in the genetically... 

Many of the amino acids originally tested by Krebs were racemic mixtures. When naturally occurring L-amino acids became available the oxidase was found to be sterically restricted to the unnatural, D series. [D-serine occurs in worms free and as D-phosphoryl lombricine (Ennor, 1959)]. It could not therefore be the enzyme used in the liver to release NH3 in amino acid metabolism. D-amino acid oxidase was shown by Warburg and Christian (1938) to be a flavoprotein with FAD as its prosthetic group. A few years later Green found an L-amino acid oxidase in liver. It was however limited in its specificity for amino acid substrates and not very active—characteristics which again precluded its central role in deamination. 

The availability of isotopes has made it possible to complete the descriptions of the steric course of most of the individual reactions of carbohydrate metabolism and steroid metabolism and many of the reactions of fat and amino acid metabolism. The subject has been covered from various angles in several chapters of the third edition of the Enzymes, particularly in the one by Popjack b, in a comprehensive treatise 2>3>, and in numerous recent reviews 4 12>. The wealth of available detail defies any attempt to be complete. I will try, rather, to describe trends in current experimentation, and to fit these trends into historical perspective. In so doing, I will select examples rather arbitrarily, entirely out of my own interests, and I beg the reader s indulgence for this bias. 

A group of enzymes which is particularly important in amino acid metabolism in the liver (and also in muscle) is the transaminases, (also called aminotransferases). These are vitamin B6 (pyridoxine) dependent enzymes which transfer an amino group from an amino acid to an oxo (keto) acid, thus ... 

There are many examples of phosphorylation/dephosphorylation control of enzymes found in carbohydrate, fat and amino acid metabolism and most are ultimately under the control of a hormone induced second messenger usually, cytosolic cyclic AMP (cAMP). PDH is one of the relatively few mitochondrial enzymes to show covalent modification control, but PDH kinase and PDH phosphatase are controlled primarily by allosteric effects of NADH, acetyl-CoA and calcium ions rather than cAMP (see Table 6.6). 

Pyridoxal phosphate is a required coenzyme for many enzyme-catalyzed reactions. Most of these reactions are associated with the metabolism of amino acids, including the decarboxylation reactions involved in the synthesis of the neurotransmitters dopamine and serotonin. In addition, pyridoxal phosphate is required for a key step in the synthesis of porphyrins, including the heme group that is an essential player in the transport of molecular oxygen by hemoglobin. Finally, pyridoxal phosphate-dependent reactions link amino acid metabolism to the citric acid cycle (chapter 16). 

The vitamin Be family of molecules are metabolic precursors to pyridoxal phosphate, an essential coenzyme for multiple enzymes involved in amino acid metabolism. 

The study of genetic defects in the oxidation of fat fuels is a relatively new field compared with the study of such defects in carbohydrate and amino acid metabolism. The first genetic defect was reported in 1970 and the first enzyme deficiency in 1973. The probable reasons for the late discovery of these defects are of some interest ... 

An intriguing puzzle in NOS catalysis is the precise role of H4B. The traditional function of H4B is in aromatic amino acid metabolism where H4B directly participates in the hydroxylation reaction via a nonheme iron. However, the NOS pterin site has no similarity to the pterin site in the hydroxylases, nor does NOS have a nonheme iron to assist pterin in substrate hydroxylation as in the amino acid hydroxylases 111). NOS more closely resembles pterin-containing enz5unes that have a redox function 81). In particular, N3 and the 03 amino group form H-bonds with either GIu or Asp residues in a series of pterin enzymes 112-116) similar to NOS, except that NOS utilizes the heme propionate (Fig. 6). 

The terminology vitamin Bg covers a number of structurally related compounds, including pyridoxal and pyridoxamine and their 5 -phosphates. Pyridoxal 5 -phosphate (PLP), in particular, acts as a coenzyme for a large number of important enzymic reactions, especially those involved in amino acid metabolism. We shall meet some of these in more detail later, e.g. transamination (see Section 15.6) and amino acid decarboxylation (see Section 15.7), but it is worth noting at this point that the biological role of PLP is absolutely dependent upon imine formation and hydrolysis. Vitamin Bg deficiency may lead to anaemia, weakness, eye, mouth, and nose lesions, and neurological changes. 

Among the NH2 transfer reactions, transaminations (1) are particularly important. They are catalyzed by transaminases, and occur in both catabolic and anabolic amino acid metabolism. During transamination, the amino group of an amino acid (amino acid 1) is transferred to a 2-oxoacid (oxoacid 2). From the amino acid, this produces a 2-oxo-acid (a), while from the original oxoacid, an amino acid is formed (b). The NH2 group is temporarily taken over by enzyme-bound pyridoxal phosphate (PLP see p. 106), which thus becomes pyridoxamine phosphate. 

The active form of vitamin Be, pyridoxai phosphate, is the most important coenzyme in the amino acid metabolism (see p. 106). Almost all conversion reactions involving amino acids require pyridoxal phosphate, including transaminations, decarboxylations, dehydrogenations, etc. Glycogen phosphory-lase, the enzyme for glycogen degradation, also contains pyridoxal phosphate as a cofactor. Vitamin Be deficiency is rare. 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

Pyridoxal phosphate is the coenzyme for the enzymic processes of transamination, racemization and decarboxylation of amino-acids, and for several other processes, such as the dehydration of serine and the synthesis of tryptophan that involve amino-acids (Braunstein, 1960). Pyridoxal itself is one of the three active forms of vitamin B6 (Rosenberg, 1945), and its biochemistry was established by 1939, in considerable part by the work of A. E. Braunstein and coworkers in Moscow (Braunstein and Kritzmann, 1947a,b,c Konikova et al 1947). Further, the requirement for the coenzyme by many of the enzymes of amino-acid metabolism had been confirmed by 1945. In addition, at that time, E. E. Snell demonstrated a model reaction (1) for transamination between pyridoxal [1] and glutamic acid, work which certainly carried with it the implication of mechanism (Snell, 1945). 

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