Substrates Phenylalanine

The structural requirements for a substrate to be reactive have been determined by measuring the values of cat and KM for a wide range of ester substrates, and the association constants of reversible inhibitors.113 The inherently high reactivity of esters causes relatively poor ester substrates to be hydrolyzed at a measurable rate. Thus esters have been most useful for working out the steric requirements of the acyl portion of the substrate. Amides and peptides are so unreactive that the only ones amenable to study are the derivatives of the specific substrates phenylalanine, tyrosine, and tryptophan. The kinetic studies may now be combined with those from x-ray diffraction. 

Figure 7. Correlation of extractable PAL activity increases with decreases in PAL substrate (phenylalanine) and products (hydroxyphenolics) during glyphosate treatment in roots of maize seedlings (39, 78),...

Substrate phenylalanine C incubation time 30 hours under 6 000 lux and 25 C agitation ... 

This points to a regulatory function of PAL in the flow of carbon from phenylalanine to phenolics derived from cinnamic acid. However, in a recent review article Margna (2) has advanced arguments that phenol accumulation in plant cells is controlled at the level of substrate (phenylalanine) supply. 

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is not considered an enzyme of the shikimate pathway proper, but since in higher plants it channels a large flux of fixed carbon into phenolic compounds, in particular lignin, the shikimate pathway must furnish its substrate, phenylalanine, in sufficient amounts. Literature on PAL has periodically been reviewed (literature in References 88 and 89) and I will concentrate here on a selective and brief discussion of our work on PAL inhibitors in the Bochum laboratory. 

PAH catalyzes the first step of phenylalanine degradation, and the reaction converts the essential amino acid phenylalanine to tyrosine. Rat liver PAH (RLPAH) has been extensively studied. Recently bacterial PAH from Chromobacterium violaceum (CVPAH) has also been studied. Because the hydroxylation is irreversible and the enzyme activity is very high in most animal liver, regulation is required. Phenylalanine and the reaction cofactor, BPH4, are regulators as well as substrates. Phenylalanine is an activator and converts the enzyme from an essentially inactive, Ei, to an active form, Ea, by binding... 

Phenylalanine hydroxylase requires molecular oxygen for its activity. Neither methylene blue nor enzymically generated hydrogen peroxide can substitute for oxygen under anaerobic conditions no activity is observed (768). However, incubation in oxygen causes some inactivation (557,768), possibly a physical effect. L-Tyrosine has been identified as a product by tracer, chromatographic, enzymic, and chemical criteria. No traces of o- or m-tyrosme can be found by methods which would detect conversion of 1% of substrate phenylalanine to either (557). 

A more interesting interaction between an enzyme and phospholipids, because of the complexity of the kinetics, is seen with phenylalanine hydroxylase from rat liver. With its naturally occurring substrates, phenylalanine and tetrahydrobiopterin, plots of v vs. [phenylalanine] are sigmoidal (Kaufman, 1971) propanol converts... 

The adaptation of the Bischler-Napieralski reaction to solid-phase synthesis has been described independently by two different groups. Meutermans reported the transformation of Merrifield resin-bound phenylalanine derivatives 32 to dihydroisoquinolines 33 in the presence of POCI3. The products 34 were liberated from the support using mixtures of HF/p-cresol. In contrast, Kunzer conducted solid-phase Bischler-Napieralski reactions on a 2-hydroxyethyl polystyrene support using the aromatic ring of the substrate 35 as a point of attachment to the resin. The cyclized products 36 were cleaved from the support by reaction with i-butylamine or n-pentylamine to afford 37. 

Carbon from the substrate glucose is converted into the carbon of the cells, phenylalanine, carbon dioxide and byproducts. Carbon balance calculations thus give us more understanding of the amount of carbon in glucose used for cell mass production, for synthesis of the wanted produd, maintenance energy and byproduct formation. 

Two reactions for the production of L-phenylalanine that can be performed particularly well in an enzyme membrane reactor (EMR) are shown in reaction 5 and 6. The recently discovered enzyme phenylalanine dehydrogenase plays an important role. As can be seen, the reactions are coenzyme dependent and the production of L-phenylalanine is by reductive animation of phenylpyruvic add. Electrons can be transported from formic add to phenylpyruvic add so that two substrates have to be used formic add and an a-keto add phenylpyruvic add (reaction 5). Also electrons can be transported from an a-hydroxy add to form phenylpyruvic add which can be aminated so that only one substrate has to be used a-hydroxy acid phenyllactic acid (reaction 6). 

Consider reaction schemes for the production of L-phenylalanine by enzymatic methods. Now match each of the following substrates with the enzyme(s) responsible for L-phenylalanine formation. 

The final conversion yield decreased when substrate concentration was increased from 2% to 4%. This was attributed to end product inhibition by the L-phenylalanine produced. Thus although faster conversion rates were observed with addition of high substrate concentrations, the product titres never exceeded 16 g l1. As already discussed the rate of yield of the conversion was proportional to the concentration of amino donor employed. Using a ratio of 1 3 substrate to amino donor, almost a 90% conversion was achieved in 3 hours. 

Assume that the cost price of L-phenylalanine produced by direct fermentation is 285 kg 1 (100 tonnes per annum capacity). What percentage reduction in substrate costs are required for 1) precursor feeding and 2) biotransformation to be competitive on a cost price basis with direct fermentation ... 

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

No compound other than the methyl ester of N-benzoyl-Lphenylalanine, 33, is an obvious choice for an open-chain analog of the locked substrate 25 but D-24, on the other hand, may be a locked analog of either N-benzoyl-D-alanine methyl ester 34 or of N-formyl-D-phenylalanine methyl ester 35 (75). If 24 is an analog of 34 rather than 35, the comparison of the two locked analogs made in Section V.B. is not valid the phenyl of 24 would then correspond to the benzoyl phenyl of 34. 

N-Benzoyl-Lalanine methyl ester is in turn about eight times more reactive than is its D enantiomer). The open-chain compounds may not bind to the enzyme in the same manner, however, as does the locked substrate. The conformation around the amido bond of the open-chain compounds, for example, can be transoid rather than cisoid (81). In addition, if equatorial 24 is considered to be the reactive conformer for both the Dand L enantiomers, and if the alanine methyl group is attracted to the hydrophobic aromatic binding subsite, then structures 34 and 38 would result. The L enantiomer of N-benzoyl-phenylalanine methyl ester 38 in this representation has approximately the same conformation as equatorial L-24. But attraction of the methyl of the D enantiomer to the location occupied by the methyl group of the L enantiomer causes the carbomethoxy group to move from the position it occupies in D-24. 

The pathway for synthesis of the catecholamines dopamine, noradrenaline and adrenaline, illustrated in Fig. 8.5, was first proposed by Hermann Blaschko in 1939 but was not confirmed until 30 years later. The amino acid /-tyrosine is the primary substrate for this pathway and its hydroxylation, by tyrosine hydroxylase (TH), to /-dihydroxyphenylalanine (/-DOPA) is followed by decarboxylation to form dopamine. These two steps take place in the cytoplasm of catecholaminereleasing neurons. Dopamine is then transported into the storage vesicles where the vesicle-bound enzyme, dopamine-p-hydroxylase (DpH), converts it to noradrenaline (see also Fig. 8.4). It is possible that /-phenylalanine can act as an alternative substrate for the pathway, being converted first to m-tyrosine and then to /-DOPA. TH can bring about both these reactions but the extent to which this happens in vivo is uncertain. In all catecholamine-releasing neurons, transmitter synthesis in the terminals greatly exceeds that in the cell bodies or axons and so it can be inferred... 

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