L-Tryptophanase

Since even a slightly elevated L-cysteine concentration is inhibitory or possibly toxic for the cells E. coli possesses another mechanism for detoxification of this compound in addition to excretion degradation of L-cysteine. Five enzymes with L-cysteine desulfhydrase activity have been identified so far in this organism L-tryptophanase (TnaA), L-cystathionine p-lyase (MetC),... 

L-tryptophan indole + pymvic acid +NH tryptophanase Proteus rettgerii ... 

FIGURE 10.3 Pathways for degradation of L-tryptophan by (a) tryptophanase, (b) deamination and oxidation, and (c) side-chain oxidation and decarboxylation to indole. 

Tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) belongs to the family of the pyridoxal 5 -phosphate (PLP)-dependent enzymes. It serves in vivo to degrade L-tryptophan, is induced by L-tryptophan, and found in various bacteria, particularly in enteric species. Tryptophanase catalyzes a,/3-elimination1 and /3-replacement reactions on interaction with L-tryptophan and various other /3-substituted amino acids2 ... 

Fig. 9.2 Crystal photographies of coli tryptophanase. A, B, and F holoenzyme, C apoenzyme, D and E complex of holoenzyme and L-alanine. See Table 9.1 for detailed, conditions.

Addition of substrates (L-tryptophan, S-methyl-L-cysteine and others) or competitive amino acid inhibitors to tryptophanase solutions induces an intense narrow absorption peak near 500 nm with a shoulder at 470-475 nm the peak rapidly disappears as the substrates are decomposed, but it is relatively stable in the presence of amino acid... 

Torchinsky et al.3S) found that the 502-nm band observed with L-alanine markedly decreases during several hours at 30° C and simultaneously pyruvate and pyridoxamine phosphate are formed. It was concluded that L-alanine undergoes slow transamination in the active site tryptophanase. 

Fig. 9.7 Effects of /3-phenyl-DL-serine, L-threonine and a-methyl-DL-serine on absorption (A) and CD (B) spectra of tryptophanase in 100 mM potassium phosphate buffer, pH 7.8, containing 2 mM dithiothreitol and 2mM EDTA. Protein concentration 1.8 mg/ml. Curve 1, unliganded enzyme. Curve 2, same as 1 + 15 mM /3-phenyl-ni -serine (threo form). Curve 3, same as 1 + 165 mM L-threonine. Curve 4, same as 1 + 500 mM a-methyl-DL-serine. (Reproduced with permission from Zakomirdina el al., Biochimie., 71, 548 549 (1989)).

A positive CD was found in the 500-nm quinonoid band which is formed on reaction of tryptophanase with L-alanine and oxindolyl-L-alanine (Fig. 9.10). The dissymmetry factor in this band is much smaller than in the absorption bands of the unliganded enzyme (Table 9.2). A negative 315-nm peak, which appears in the presence of L-alanine (Fig. 9.10), may be caused by interaction of an aromatic amino acid residue with the quinonoid coenzyme ring. 

Downfield H-NMR spectra of tryptophanase in H2O were measured for studying hydrogen-bonded network around PLP in the active site.49 The peaks at 17.7 and 16.3 ppm observed in the presence of L-threonine and L-alanine, respectively, were tentatively ascribed to the proton on the ring nitrogen of PLP. 

Nihira, Toraya and Fukui53 found that irradiation of holotryptophanase solutions with visible light led to rapid, pH-dependent loss of enzymatic activity. The apoenzyme was not inactivated under the same conditions therefore, the inactivation was PLP-sensitized. L-Tryptophan and l-alanine markedly decreased the rate of photoinactivation. From a plot of the inactivation rate versus pH, the pKa was found to be 7.2, which is close to the pKa of the imidazole group. Photoinactivation was accompanied by destruction of one histidine residue per subunit. No concomitant loss of tryptophan, tyrosine or methionine was detected, and it was suggested that tryptophanase contains an essential histidine residue near the active site. 

The presence of an essential tyrosine residue near the active site was suggested on the basis of experiments with tetranitromethane.54 Treatment of apotryptophanase with this reagent caused almost complete loss of catalytic activity, a great reduction of affinity for PLP and modification of about one tyrosine residue. The modified enzyme was unable to form the quinonoid intermediate with L-tryptophan or L-alanine.55 PLP protected the apoenzyme from inactivation only in the presence of activating cations (K+, NH4+, Rb+). It was shown that inactivation by tetranitromethane was not caused by oxidation of SH-groups, but partial modification of methionine (0.8 residue) was detected and might also be responsible for inactivation. It is worthy of note that modification of tryptophanase with chloramine T indicated that some methionine residues may be important for maintaining the catalytically active conformation of the enzyme.56 ... 

It has been found68-70 that the reaction of tryptophanase with the inhibitory amino acids, /3-phenyl-DL-serine (threo form), L-threonine and D-alanine, is accompanied by a manifold increase in the reduced LD, i.e. in the ratio of LD to absorbance (AA / A) in the 420-425 nm band (Fig. 9.12 Table 9.2). This band belongs to the protonated internal PLP-lysine... 

In the active site of the enzyme PLP forms an internal aldimine (Schiff base) with Lys-270 (Fig. 9.13,1). When the substrate is bound at the active site, its a-amino group attacks the C-4 atom of the coenzyme and replaces the -amino group of Lys-270 from its bond with PLP. This transaldimination reaction probably proceeds via a tetrahedral intermediate (a gem-diamine). Spectral evidence for formation of a gem-diamine in this step has recently been obtained in studies of the reaction of tryptophanase with L-homophenylalanine.41 The gem-diamine is subsequently converted to an external, PLP-substrate aldimine, and the -amino group of Lys-270 is released (Fig. 9.13, II). The equilibrium constant of this step with L-tryptophan is determined to be 11.6 mM.78 ... 

Furthermore, with the addition of tryptophanase, an enzyme that selectively digests tryptophan, the sperm failed to navigate toward live eggs. Thus, a natural gradient of L-tryptophan is considered to be essential. 

The closely related bacterial enzyme tyrosine phenol-lyase [137] has an even wider substrate and reaction specificity than tryptophanase, including the remarkable ability to cleave both D- and L-tyrosine and to interconvert D- and L-alanine. As already discussed and summarized in Tables 1 and 2, the stereochemistry at C-/J in all the a,/3-elimination and -replacement reactions of this enzyme studied so far is always retention [108,109,129]. This includes the a, -elimination of L- as well as of D-tyrosine. The fate of the a-hydrogen of L-tyrosine in this reaction has been probed in preliminary experiments (H. Kumagai, E. Schleicher and H.G. Floss, unpublished results), and the results tentatively suggest transfer of deuterium from the a-position to C-4 of the resulting phenol. Attempts to demonstrate intramolecularity of this transfer have so far been inconclusive. The base abstracting H-a in this enzyme may be histidine [138]. 

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