Tyrosine formation

Histone HI from the fruit fly Ceratitis capitata has two tyrosine residues. Jordano et al.(<)2> have observed two differences from calf thymus HI (1) the apparent quantum yield does not increase on protein folding and (2) there is a pH- and conformation-dependent shoulder at 340 nm in the emission spectrum. This group has attributed this 340-nm emission to tyrosinate.(97) Their studies demonstrate that the folding of histone HI from C. capitata is pH and ionic strength dependent. The possibility of tyrosinate formation at neutral pH is discussed in greater detail in Section 1.5.2. 

An additional emission band near 350 nm has been observed for lima bean trypsin inhibitor (LBTI).(173) The authors discussed both the possibility of contamination by tryptophan and excited-state tyrosinate formation. Since this 350-nm emission has a tyrosine-like excitation spectrum that is slightly shifted compared to that of the major 302-nm emission, it is also possible that the tyrosine residue in a fraction of the LBTI molecules could be hydrogen bonded. This model is supported by the observations that the phenol side chain is shielded from solvent and has an anomalously high pKa. 

Table 4 lists some representative examples of macrocyclization via the intramolecular SNAr reaction. In addition to the 14- and 17-membered cycloisodityrosines shown in the table, a variety of mono-, bi-, and tricyclic systems from the vancomycin family of natural products including the orienticin C/40 vancomycin J44-47 and teicoplanin[48 aglycons have been prepared by this method.This versatility, coupled with the high yields obtained, makes the intramolecular SNAr reaction currently the most widely applicable method for cycloisodi-tyrosine formation. 

Phenylalanine hydroxylase mononuclear iron tyrosine formation 9 3.2.2. 

Scheme 15 Mechanism for tyrosine formation from phenylalanine following hydroxyl radical attack...

Biochemistry of Phenylalanine and Tyrosine Formation in Vascular Plants The Entry Point to Phenylpropanoid Metabolism and to Lignification... 

Support for the argument that the cofactor is the site for oxygen activation stems from two sets of experiments. Kaufman [105] first observed the accumulation of a transient species during PAH turnover that decays non-enzymatically or, in the presence of a stimulator protein, enzymatically to quinonoid-BPHj. Since the rate of tyrosine formation was more rapid than decay of this species, the transfer of oxygen must already be complete. This deduction led to the postulate that the intermediate is comprised of elements from both BPH4 and the remaining oxygen... 

CYP56 C-C coupling ofN-formyl tyrosine Formation of N, N -bisformyl dityrosine for outer spore wall production E.g., C. albicans, S. cerevisiae [455 57, 832]... 

It is interesting that Udenfriend and Cooper (J.86) had found that an organic alcohol or aldehyde was required in the enzyme system for the oxidized diphosphopyridine to function and that Mitoma ef of. 191) demonstrated that this was required for the reduction of the pyridine nucleotide. In this connection it is to be noted that glucose dehydrogenase stimulates tyrosine formation in the purified enzyme i ystem of Kaufman 190) in the absence of glucose and in the presence of a large excess of TPNH. This enzyme is included in the incubation medium employed by Kaufman. 

Work on this problem was initiated in the author s laboratory in 1955. When extracts of livers of various animals were assayed for tyrosine formation, it was found that rat liver extracts had the highest activity many of the extracts, such as those prepared from sheep liver, were almost completely inactive. This inactivity was quite surprising since all mammalian livers can presumably convert phenylalanine to tyrosine. Subsequently (Kaufman, 1959) it was found that sheep liver homogenates, in contrast to extracts, had good activity, indicating that some component of the hydroxylating system in sheep liver is not readily solubilized. It is not known if this is the general explanation for the inactivity or low activity of some of the other mammalian liver extracts which were assayed. 

TPNH oxidation in the presence of phenylalanine is compared to that in its absence. Just as with tyrosine formation, the phenylalanine-dependent oxidation of TPNH required the presence of both the rat and sheep liver enzymes. 

As can be seen in Fig. 1, there was a pronounced lag period before the rate of TPNH oxidation in the presence of phenylalanine exceeded that of the control. This phenomenon was studied in some detail because it was felt that it might provide a clue to the first step in the sequence of reactions which leads to tyrosine formation. It was found that a short anaerobic preincubation of the sheep liver enzyme with TPNH eliminated the lag period almost completely (Kaufman, 1958a). In addition, it was found that the lag period could be restored if the anaerobic preincubation was followed by a brief exposure to air. These findings suggested that during the lag period, some cofactor present as a contaminant in the partially purified sheep liver enzyme was reduced to an active form by TPNH and that this reductive reaction was catalyzed by the sheep liver enzyme. The restoration of the lag period by exposure to air indicated that the reduced compound was autoxidizable, as shown by Eqs. 4, 5, and 6, where X stands for the postulated cofactor. 

Fig. 2. Stimulation of tyrosine formation by the cofactor isolated from rat liver.

Fig. 3. Comparison of the time course of tyrosine formation with tetrahydrofolate and the cofactor. 0> 0-12 /nmoles of tetrahydrofolate , 10 /ng. cofactor.

A comparison of the requirements of the system when the rat liver cofactor and the dimethyltetrahydropteridine are used is shown in Table I (Kaufman, 1959). With either compound, tyrosine formation showed an absolute requirement for oxygen, phenylalanine, and the rat liver enzyme. It can be seen, however, that, in sharp contrast to the results obtained with the natural cofactor, the requirement for TPNH and sheep liver enzyme was no longer absolute in the presence of the dimethyltetrahydropteridine. This represented the first demonstration of tyrosine formation in their complete absence. This result provided convincing evidence in favor of the idea that neither TPNH nor the sheep liver enzyme was directly involved in the hydroxylation reaction, an idea already postulated from the results of studies on the lag period in the phenylalanine-stimulated oxidation of TPNH. 

Dependence of Tyrosine Formation on Sheep Liver Enzyme at Varying Concentrations of TPNH and 2-Amino-4-hydroxy-6,7-dimethyltetrahy-... 

The final point to be made about the data shown in Fig. 6 is that the initial rate of tyrosine formation was independent of the presence of TPNH. Furthermore, although not shown in the figure, the initial rate of the reaction was independent of sheep liver enzyme. 

Fig. 6. Time course of tyrosine formation in the presence and absence of TPNH. 0.12 /imoles of tetrahydropteridine used.

If an experiment similar to the one described in Fig. 6 was carried out and TPNH was added after the reaction had been allowed to proceed for 15 minutes, little additional tyrosine formation could be detected on subsequent incubation (curve D, Fig. 7). Under these conditions the system had become inactive. Phenylalanine was required for this almost complete loss in activity because a control incubation carried out in the absence of phenylalanine led to only a small decrease in the amount of tyrosine which could be formed during a second incubation with phenylalanine. When a second addition of tetrahydropteridine or of both TPNH and tetrahydropteridine was made after 15 minutes (curves C and B, respectively), the system was fully active, demonstrating that the loss in activity was due neither to inactivation of one of the enzymes nor to the formation of an inhibitor. 

Pig. 7. The effect of second additions of the cofactors on the rate of tyrosine formation. 

An assay for the detection of the intermediate was divised which is based on an experiment similar to the one described in Fig. 7 where the conversion of phenylalanine to tyrosine was allowed to proceed in the absence of TPNH. When there was no further tyrosine formation, TPNH was added and a second incubation was carried out. Any additional tyrosine formation during this second incubation indicated that a compound had been formed during the first incubation which could be reduced to an active tetrahydropteridine in the presence of TPNH. 

When this experiment was carried out under the conditions of the standard assay, using the dimethyltetrahydropteridine, there was no evidence for the accumulation of an active compound (see Fig. 7). When Tris buffer was substituted for the phosphate buffer ordinarily used in the standard assay, an active compound accumulated, as shown in Fig. 8 (Kaufman, 1959). In the absence of TPNH, tyrosine formation... 

Fig. 8. The effect of addition of TPNH after tyrosine formation was stopped. Tris buffer used in place of phosphate buffer.

Fig. 9. It is apparent that even under these conditions the compound is quite unstable after 45 minutes, there was very little tyrosine formation after the addition of TPNH (curve F). Several unsuccessful attempts were made to isolate the intermediate from reaction mixtures. In every...

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