Phenylpyruvate transamination

Figure 2. Alternative enzymatic routing for L-phenylalanine biosynthesis. Dehydration followed by transamination defines the phenylpyruvate route, whereas the reverse order of reactions defines the arogenate route. Abbreviations GLU, L-glutamate aKG, 2-ketoglutarate.

Although 2-phenylethanol can be synthesised by normal microbial metabolism, the final concentrations in the culture broth of selected microorganisms generally remain very low [110, 111] therefore, de novo synthesis cannot be a strategy for an economically viable bioprocesses. Nevertheless, the microbial production of 2-phenylethanol can be greatly increased by adding the amino acid L-phenylalanine to the medium. The commonly accepted route from l-phenylalanine to 2-phenylethanol in yeasts is by transamination of the amino acid to phenylpyruvate, decarboxylation to phenylacetaldehyde and reduction to the alcohol, first described by Ehrlich [112] and named after him (Scheme 23.8). 

In this transamination, the effect of para substitient groups has been studied using fluorinated phenylpyruvic acids and L-aspartic acid. From these results, the migratory preference is H > F > Cl > Br > CF3. This order has been attributed to the bulkiness of the substituted group [57]. Direct amination of p-substituted succinic acid with phenylalanine ammonialyase (EC 4.3.1.5) has suggested very high substrate specificity that the order of reaction rate is m-F o-F P-p-F >CF3. 

In individuals with PKU, a secondary, normally little-used pathway of phenylalanine metabolism comes into play. In this pathway phenylalanine undergoes transamination with pyruvate to yield phenylpyruvate (Fig. 18-25). Phenylalanine and phenylpyruvate accumulate in the blood and tissues and are excreted in the urine—hence the name phenylketonuria. Much of the phenylpyruvate, rather than being excreted as such, is either decarboxylated to phenylacetate or reduced to phenyllactate. Phenylacetate imparts a characteristic odor to the urine, which nurses have traditionally used to detect PKU in infants. The accumulation of phenylalanine or its metabolites in early life impairs normal development of the brain, causing severe mental retardation. This may be caused by excess phenylalanine competing with other amino acids for transport across the blood-brain barrier, resulting in a deficit of required metabolites. 

Figure 25-5 shows the principal catabolic pathways, as well as a few biosynthetic reactions, of phenylalanine and tyrosine in animals. Transamination to phenylpyruvate (reaction a) occurs readily, and the product may be oxidatively decarboxylated to phen-ylacetate. The latter may be excreted after conjugation with glycine (as in Knoop s experiments in which phenylacetate was excreted by dogs after conjugation with glycine, Box 10-A). Although it does exist, this degradative pathway for phenylalanine must be of limited importance in humans, for an excess of phenylalanine is toxic unless it can be oxidized to tyrosine (reaction b, Fig. 25-5). Formation of phenylpyruvate may have some function in animals. The enzyme phenylpyruvate tautomerase, which catalyzes interconversion of enol and oxo isomers of its substrate, is also an important immunoregulatory cytokine known as macrophage migration inhibitory factor.863...

In cases where the natural amino acid side chains of enzymes are insufficient to carry out a desired reaction, the enzyme frequently uses coenzymes. A coenzyme is bound by the enzyme along with the substrate, and the enzyme catalyses the bimolecular reaction between the coenzyme and the substrate (cf. Section 2.6.3). A simple model for a-amino acid synthesis by transamination was developed by substituting /I-cyclodextrin with pyridoxamine. Pyridoxamine is able to carry out the transformation of a-keto acids to a-amino acids even without the presence of the cyclodextrin, but with the cyclodextrin cavity as well, the enzyme model proves to be more selective and transaminates substrates with aryl rings bound strongly by the cyclodextrin much more rapidly than those having little affinity for the cyclodextrin. Thus (p-le/f-butylphenyl) pyruvic acid and phenylpyruvic acid are transaminated respectively 15 000 and 100 times faster then pyruvic acid itself, to give (p-le/f-butylphenyl) alanine and phenylalanine (Scheme 12.5). 

An inability to degrade amino acids causes many genetic diseases in humans. These diseases include phenylketonuria (PKU), which results from an inability to convert phenylalanine to tyrosine. The phenylalanine is instead transaminated to phenylpyruvic acid, which is excreted in the urine, although not fast enough to prevent harm. PKU was formerly a major cause of severe mental retardation. Now, however, public health laboratories screen the urine of every newborn child in the United States for the presence of phenylpyru-vate, and place children with the genetic disease on a synthetic low-phenylalanine diet to prevent neurological damage. 

They lack the enzyme phenylalanine oxidase, which converts phenylalanine to tyrosine. Thus phenylalanine accumulates in the body and it is degraded to phenylpyruvate by transamination ... 

Murakami et al. also found that the transamination reaction between hydrophobic pyridoxals (36 and 37) and a-amino acids, to produce a-keto acids, was extremely slow for neutral pyridoxals even in the presence of Cu(n) ions [24]. Detailed kinetic analysis of the reactions carried out in the vesicular system indicated that the transformation of the Cu(n) -quinonoid chelate into the Cu(n) -ketimine chelate was kinetically unfavorable compared with the competing formation of the Cu(n)-aldimine chelate from the same quinonoid species. This problem was solved to a certain extent by quaternization of the pyridyl nitrogen in pyridoxal, as Murakami et al. successfully accomplished transamination between catalyst 36 and L-phenylalanine to produce phenylpyruvic acid. 

To study the effect of polymer size on catalysis [37], pyridoxamine was linked to a series of PEIs with Mn = 600,1800,10 000, and 60 000, both simply permethylated and with additional attached dodecyl chains. The polymers were examined in the transamination of pyruvic acid and of phenylpyruvic acid, showing Michaelis-Menten behavior. The k2 and of i M determined showed only small variations with polymer size. Thus, the strong advantage of pyridoxamines attached to the Mn = 60 000 PEI, relative to simple pyridoxamine alone, was seen to almost the same extent with the smaller... 

As illustrated in Figure 19-1, there are two different competing pathways available for the catabolism of PA. One pathway involves transamination of PA to phenylpyruvate, followed by the phenylpyruvate dehydrogenase reaction to form phenylacetyl-CoA. The products of these reactions (phenylpyruvate, phenylacetyl-CoA) and the excretion products phenylacetate and phenyllactate cannot be further metabolized and consequently accumulate in the blood, urine, and tissues. 

Phenylketonuria is due to an inborn error of phenylalanine metabolism. Typically, it is due to a deficiency of phenylalanine hydroxylase. Atypically, it can be caused by a deficiency of dihydrobiopterin reductase and a resultant inability to synthesize biopterin. All these conditions cause an accumulation of phenylalanine, which can be transaminated to phenylpyruvic acid. 

We have arrived at prephenic acid, which as its name suggests is the last compound before aromatic compounds are formed, and we may call this the end of the shikimic acid pathway. The final stages of the formation of phenylalanine and tyrosine start with aromatization. Prephenic acid is unstable and loses water and CO2 to form phenylpyruvic acid. This a-keto-acid can be converted into the amino acid by the usual transamination with pyridoxal. 

This compound has been used as a chemical model for pyridoxamine. For example, it transaminates phenylpyruvate under the conditions shown here. Comment on the analogy and the role of Zn(II). In what ways is the model compound worse and in what ways better than pyridoxamine itself ... 

The pathway bifurcates at chorismate. Let us first follow the prephenate branch (Figure 24,17). A mutase converts chorismate into prephenate, the immediate precursor of the aromatic ring of phenylalanine and tyrosine. This fascinating conversion is a rare example of an electrocyclic reaction in biochemistry, mechanistically similar to the well-known Diels-Alder reaction from organic chemistry. Dehydration and decarboxylation yield phenylpyruvate. Alternatively, prephenate can be oxidatively decarboxylated to p-hydroxyphenylpyruvate. These a-ketoacids are then transaminated to form phenylalanine and tyrosine. 

The production of L-phenylalanine from the precursor phenylpyruvic acid by transamination is a process which requires two steps ... 

Although the phenylalanine-derived fragment of cytochalasin D (148) possesses the configuration of the L-amino-acid both D- and L-phenylalanine are equally effective precursors. Incorporation occurs with complete loss of tritium and N from C-2 and extensive loss of tritium from both diastereotopic hydrogens at C-3. These losses could be explained as occurring in part during the course of transamination and phenylpyruvic acid may then be implicated. This acid depressed the incorporation of D-phenylalanine as measured relative to the L-isomer (2S)-[4 - H]- and (2/ 5)-[2- C]-phenylalanine were fed the cytochalasin isolated showed an increased H C ratio, cf. discussion on p. 1. It follows that L-phenylalanine rather than the D-isomer is the primary precursor of cytochalasin D (148). 

Phenylketonuria (PKU) is an inborn error of metabolism by which the body is unable to convert surplus phenylalanine (PA) to tyrosine for use in the biosynthesis of, for example, thyroxine, adrenaline and noradrenaline. This results from a deficiency in the liver enzyme phenylalanine 4-mono-oxygenase (phenylalanine hydroxylase). A secondary metabolic pathway comes into play in which there is a transamination reaction between PA and a-keto-glutaric acid to produce phenylpyruvic acid (PPVA), a ketone and glutamic acid. Overall, PKU may be defined as a genetic defect in PA metabolism such that there are elevated levels of both PA and PPVA in blood and excessive excretion of PPVA (Fig. 25.7). 

D-Amino acids vary in availability with the species. For example d-phenylalanine is used by rat, mouse, and man (15, 35, 727, 730, 962), whereas D-tryptophan is used by the rat (53, 54, 759, 895), is partially used by the mouse and pig (139, 867), and is not used by man (7, 29). The utilization of the D-amino acids is probably determined by the relative rates of absorption of the D-amino acid from the intestine, and of conversion of d- to L-amino acid in the liver (288). The conversion of d- to L-phenylalanine is reduced in vitamin-Be deficiency (52), as is to be expected for a transformation involving transamination to phenylpyruvic acid. Phenylpyruvic and indolepyruvic acids, the a-keto acids corresponding to phenylalanine and tryptophan, may also, to an extent varying with the species, satisfy growTh requirements (e.g., 55, 109, 436, 725, 911). 

Phenylketonuria The presence of elevated amounts of phenylketones, primarily phenylpyruvate, in urine a primary indication of disturbance of phenylalanine metabolism resulting from elevated transamination of phenylalanine due to reduction in phenylalanine hydioxylalion to tyrosine. 

Figure 38-1. Transamination of phenylalanine to yield phenylpyruvate, a phenylketone.

The provision of reducing equivalents to phenylalanine hydroxylase is dependent on reduction of dihydrobiopterin by NADH catalyzed by the enzyme dihydropteridine reductase, as shown in Figure 38-2. This reduction is dependent on the availability of biopterin and therefore on the biopterin synthetic pathway. Thus any genetic or protein folding defect in either dihydropteridine reductase or the biopterin biosynthetic enzymes would compromise the efficacy of phenylalanine hydroxylation to tyrosine resulting in hyperphenylalaninemia and also phenylketonuria resulting from inaease transamination of phenylalanine to phenylpyruvate. 

Phenylketones Normally minor metabolites of phenylalanine that result from the transamination of phenylalanine and further reduction. These include phenylpyruvate and phenyllactate. These metabolites are elevated when the conversion of phenylalanine to tyrosine is impaired. 

Because transamination reactions are reversible, it is theoretically possible for all amino acids to be synthesized by transamination. However, experimental evidence indicates that there is no net synthesis of an amino acid if its a-keto acid precursor is not independently synthesized by the organism. For example, alanine, aspartate, and glutamate are nonessential for animals because their a-keto acid precursors (i.e., pyruvate, oxaloacetate, and a-ketoglutarate) are readily available metabolic intermediates. Because the reaction pathways for synthesizing molecules such as phenylpyruvate, a-keto-/Thydroxybutyrate, and imidazolepyruvate do not occur in animal cells, phenylalanine, threonine, and histidine must be provided in the diet. (Reaction pathways that synthesize amino acids from metabolic intermediates, not only by transamination, are referred to as de novo pathways.)... 

Phenylketonuria, caused by a deficiency of phenylalanine hydroxylase, is one of the most common genetic diseases associated with amino acid metabolism. If this condition is not identified and treated immediately after birth, mental retardation and other forms of irreversible brain damage occur. This damage results mostly from the accumulation of phenylalanine. (The actual mechanism of the damage is not understood.) When it is present in excess, phenylalanine undergoes transamination to form phenylpyruvate, which is also converted to phenyllactate and phenyl-acetate. Large amounts of these molecules are excreted in the urine. Phenylacetate gives the urine its characteristic musty odor. Phenylketonuria is treated with a low-phenylalanine diet. 

Table 12.7-1. Time course for the transamination of phenylpyruvate to L-phenylalanine in the presence and absence of oxaloacetate decarboxylase.

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