Pyridoxal phosphate tyrosine oxidation

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

The experiments described earlier showed that in liver homogenates and extracts this reaction is brought about by transamination, which is an obligatory first step in the oxidation of tyrosine by such systems. The existence of su( h a transaminating system was already known (133, 134, 393), and the observed pyridoxal phosphate-dependence when transamination was was made rate-controlling (489) was in accordance with the known behavior of transaminases (c/. 482). 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Prephenate (26) is converted to phenylpyruvate (28) in many bacterial systems. In some organisms, such as Escherichia coli, prephenic acid also is oxidatively aromatized to / -hydroxyphenylpyruvic acid (29) by a soluble, NAD+-de-pendent enzyme, prephenate dehydrogenase. p-Hydroxy-phenylpyruvic acid is then transaminated by the addition of L-glutamate and pyridoxal phosphate to yield L-tyrosine (30). 

The first step in tyrosine oxidation is a transamination to form p-hydroxyphenylpyruvic acid. Several groups of investigators independently showed a dependence of tyrosine oxidation on the presence of a keto acid. Knox and LeMay-Knox showed that a-ketoglutarate is a specific partner in the transamination and that pyridoxal phosphate is a cofactor in this reaction. Partial resolution of the transaminase allowed a demonstration of parallel restoration of transaminase activity and over-all tyrosine oxidation by addition of pyridoxal phosphate. 

A kochsaft of liver or yeast has an accelerating effect on tyrosine oxidation by acetone powder extracts of liver. This suggests a requirement for an unknown coenzyme. Tests carried out with known eoenzymes revealed that, in addition to pyridoxal phosphate, coenz3nme A on occasions had a stimulatory effect and that this coenzyme partly reversed the inhibitory effect of pyruvate. 

In bacteria and other micro-organisms, unlike higher organisms, L-phenylalanine (1) is not normally a precursor of L-tyrosine (2). Davis postulate that in these organisms prephenic acid is the precursor of L-tyrosine (2) and subsequent work at the enzymic level by Schwink and Adams established this proposal. Thus in Escherichia coli prephenic acid (31) is oxidatively aromatised to p-hydroxyphenylpyruvic acid (33) by a soluble, NAD" dependent, enzyme—prephenate dehydrogenase. With appropriate fortification —addition of L-glutamate and pyridoxal phosphate as cofactors —extracts of Escherichia coli then convert p-hydroxyphenylpyruvic acid quantitatively to L-tyrosine by transamination. 

The indirect evidence for the transamination is that only four atoms of oxygen are required to oxidize one molecule of tyrosine to acetoacetate, a-ketoglutarate stimulates the oxidation, no free ammonia is produced in the reaction, the oxidation is decreased upon removal of the transaminase coenzyme by ammonium sulfate precipitation or by dialysis, and there is an increase in the rate of tyrosine oxidation upon addition of pyridoxal phosphate. When p-hydroxyphenylpyruvate was used as the substrate addition of keto acids did not increase the rate of oxidation. 

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