Tryptophan pyrrolase reaction

This paper describes a summary of our recent studies on the mechanism of the tryptophan pyrrolase reaction. Particular emphasis is placed on the role of heme as oxygen binding site. 

Oxygenation and hydroxylation of a wide variety of biological materials almost always involves the participation of a metal ion, usually iron and sometimes copper. In one unique case, tryptophane pyrrolase, the iron is present as heme (75). The only dioxygenase enzyme reaction in which a metal ion has not been implicated is one involved in the degradation of vitamin B6 (16). 

The focus of this chapter is the reaction site of oxygenases (hemo-proteins) having heme as the prosthetic group. We discuss the oxygen activation in tryptophan pyrrolase (TPO) and cytochrome P-450 based on our experimental results using iron-porphyrin complexes as the model for the active site of these enzymes. 

Tryptophan Pyrrolase Model Reaction and Activation of Oxygen... 

Tryptophan is an essential amino acid involved in synthesis of several important compounds. Nicotinic acid (amide), a vitamin required in the synthesis of NAD+ and NADP+, can be synthesized from tryptophan (Figure 17-24). About 60 mg of tryptophan can give rise to 1 mg of nicotinamide. The synthesis begins with conversion of tryptophan to N-formylkynurenine by tryptophan pyrrolase, an inducible iron-porphyrin enzyme of liver. N-Formylkynurenine is converted to kynurenine by removal of formate, which enters the one-carbon pool. Kynurenine is hydroxylated to 3-hydroxykynurenine, which is converted to 3-hydroxyanthranilate, catalyzed by kynureninase, a pyridoxal phosphate-dependent enzyme. 3-Hydroxyanthranilate is then converted by a series of reactions to nicotinamide ribotide, the immedi-... 

Figure 4, Oxygenated form of tryptophan pyrrolase. Spectrum was recorded during the continuous bubbling of oxygen to the reaction mixture at S C. Reaction mixture contained ferrous tryptophan pyrrolase (specific activity 3.7), 11.4 mg. lu-tryptophan, 50 ujnoles potassium phosphate buffer, pH 7.0, 500 fxmoles, in a final volume of 5.0 ml.

Figure 5. Conversion of the ferric enzyme to cyanide complex in the presence and absence of tryptophan. Reaction mixture contained tryptophan pyrrolase, 0.47 mg. (specific activity 2.5) potassium phosphate buffer, pH 7.0, 1.1. mmoles i.-tryptophan, 0.28 mmoles in a jfinal volume of 28 ml. Cyanide was added in the presence (O) and absence ( X) of tryptophan as indicated...

The mechanism of hydroxylation of aromatic compounds catalyzed by peroxidase in the presence of DHF and Oo has been discussed by Mason et al. (i, 14, 15). Probably, the activated species of O2 is per-hydroxyl ion in this reaction (I), but the possible transfer of an oxygen atom from III to the organic molecule as suggested by Mason, may not be excluded (15). The similarity of this enzyme to tryptophan pyrrolase has been discussed. Now it is found that tryptophan pyrrolase shows... 

Laborious studies in different laboratories elucidated the pathway used by tryptophan to yield nicotinic acid. The intermediates are outlined in the metabolic map (see Fig. 4-9). The first of these reactions is catalyzed by tryptophan pyrrolase, a heme-contain-... 

The first of the enzymes catalyzing this sequence of reactions is now named tryptophan pyrrolase. The reaction promoted is given in Eq. (19). 

Formation of kynurenine from tryptophan was discovered in liver extracts by Kotake and Masayama 289). These authors proposed the name tryptophan pyrrolase for the enzyme. Knox and Mehler 290) subsequently showed that the formation of kynurenine consisted of two enzyme reactions, an initial oxidation to formylkynurenine followed by hydrolysis to kynurenine. Because the reaction was stimulated by HsO produced in situ it was assumed that there was an intermediate formation and utilization of peroxide in the oxidation. For this reason the enzyme was renamed tryptophan oxidase-peroxidase by Knox. Experiments with O showed that molecular O2 was incorporated into the reaction products 291). One mole of O2 per mole of tryptophan was contained in the formylkynurenine. When H20 was tested, very little 0 was utilized. This observation led Tanaka and Knox 292) to return to the use of the original name, tryptophan pyrrolase. 

Neurospora mutants. Kotake and collaborators (35) obtained evidence for the conversion of tryptophan to formylkynurenine in cat and dog liver extracts the enzyme carrying out this activity was named tryptophan pyrrolase. The oxidation of tryptophan to formylkynurenine [reaction (I), Fig. 1 ] appeared to take place in two stages. Knox and Mehler (36, 37), in careful studies with mammalian liver enzymes, showed that the oxidation required both molecular oxygen and hydrogen peroxide dyes cannot substitute in this reaction. It has been postulated that the two stages involve (a) the formation of an intermediate resulting from the action of peroxide on tryptophan as indicated by the following equation... 

Tanaka and Knox (40) have recently found that peroxide is not directly involved in the formation of formylk3Tiurenine, but acts in converting an inactive ferric enzyme in the presence of tryptophan into an active ferrous protein. It is of interest that cyanide and catalase inhibit the inactive enzyme but not the active ferrous form. On the other hand, carbon monoxide inhibits only the active enzyme. The changes have been summarized by Tanaka and Knox according to the scheme given in Fig. 3. These authors have suggested, since the reaction involves a direct oxygenation of the substrate, that the enzyme be termed tryptophan pyrrolase rather than the previously used nomenclature of tryptophan-peroxidase-oxidase. 

Fig. 24. Activation of tryptophan pyrrolase by RNAase T,. Drosophila melanogaster were homogenized in 4 vol of 0.05 M potassium phosphate (pH 7.5) per g of frozen adult flies and centrifuged at 15,000 g for 30 min. The tryptophan pyrrolase was assayed by using a modified Bratton-Marshal assay to detect kynurenine production that results from adding 0.1 ml of homogenate to 1 ml of assay mixture consisting of 0.005 M L-tryptophan, 0.001 M 2-mercaptoethanol, 0.1 M potassium phosphate (pH 7.5) and oxygen at a concentration that results from equilibration with air. The reaction occurred at 37 °C and was stopped with 0.33 ml of 20% trichloroacetic acid at the times indicated. RNAase T, was either present at 2.5 U/ml...

Tryptophan oxygenase (tryptophan pyrrolase) plays an important role in the metabolism of tryptophan and has been prepared from animal tissues (Knox and Mehler, 1950) and bacteria (Hayaishi and Stanier, 1951). Enzymes from the two sources were found to be comparable in many respects. Knox and Mehler named the enzyme tryptophan peroxidase-oxidase, since they had found that catalase inhibits the reaction and that this inhibition is reversed by hydrogen peroxide, suggesting the intermediate formation and the subsequent utilization of peroxide as shown in Eqs. (21) and (22). 

A third pathway of protocatechuic acid metabolism occurs in a Rhodo-pseudomonas. Proctor and Scher (1960) have reported that this organism forms protocatechuate from benzoate, then decarboxylates the product to catechol. (This is the second example of a nonoxidative aromatic decarboxylase.) Subsequent metabolism of catechol yields a keto acid not yet identified, but possibly that produced in other catechol-utilizing systems described below. A unique feature of the Rhodopseudomonas system is its reported dependence on hydrogen peroxide for the oxidation of catechol. It will be of interest to learn whether peroxide is consumed stoichiometrically in the reaction, or whether it is an activator, as has been found for tryptophan pyrrolase. 

The tryptophan-oxidizing activity can be assayed by various determinations of the substrate, but more rapid and convenient assays have been used based on the appearance of the product. The initial product of tryptophan oxidation is A -formylkynurenine, but this is rapidly hydrolyzed by an enzyme, kynurenine formylase (Mehler and Knox, 1951) or formamidase, that is much more active than tryptophan pyrrolase. The kynurenine that is produced in the second reaction accumulates... 

The inhibitors of tryptophan pyrrolase may be classified into those that prevent activation of the enzyme and those that inhibit the active form. Catalase, as mentioned earlier, was required in certain amounts to balance peroxide-generating systems, but inhibited in higher concentrations. These effects have now been shown to concern only the activation process, and catalase has been found to have no influence on the activated tryptophan pyrrolase (Tanaka and Knox, 1959). Similarly, peroxides are required for the activation, but cause irreversible inactivation if added in the absence of tryptophan. Cyanide is a potent inhibitor if added before activation, but has very little effect if added during the reaction. Ferricyanide causes almost complete cessation of activity when added during the reaction, as was expected if only the ferrous form of the enzyme were active. This interpretation is supported by the reactivation of ferricyanide-treated enzyme by ascorbic acid, which presumably reduces the iron back to the ferrous state. However, if cyanide is added before the ascorbic acid, there is very little reactivation. Carbon monoxide causes an inhibition that is reversed by light. All of these observations are consistent with a model in which an inactive ferric enzyme is reduced to an active form by peroxide and tryptophan. The ferric form combines readily with cyanide (and also with azide and hydroxylamine), while the ferrous form combines with carbon monoxide. 

The evidence from Suda s laboratory indicates that in pyrocatechase and homogentisic oxidase the iron is bound to a tyrosine residue. In tryptophan pyrrolase the iron is present as heme. In the case of reactions involving bonds adjacent to a catechol or o-amino phenol group, the hydroxyl or amino group away from the bond to be attacked may participate in the iron complex. In all cases the iron may be assumed to be bound in a complex that permits very little dissociation (as measured by exchange with free ferrous ions) at neutral or alkaline pH values. 

---