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PubMed1975

[Differentiation of tryptophanases of five species of Enterobacteriaceae by sulfhydryl groups].

We have studied the sulfhydryl groups (-SH) on the tryptophanases (TPases) from Escherichia coli B. and E. aurescens, Shigella alkalescens, and Proteus vulgaris and P. morganii. The coli group and the P. morganii apo TPases have 20 -SH groups per mole of enzyme, where as P. vulgaris apoTPase has 16. In coli group TPases, there are 16 -SH groups on the mole surface and they are all implicated in the activity and the enzyme-substrate bond. Proteus morganii TPase has 8 surface -SH groups, 4 of which are implicated in the activity; the remaining 12 -SH groups are located inside the mole and take part in the activity and the enzyme-substrate bond. Proteus vulgaris TPase has 4 surface -SH groups which are constructive of the enzyme structure, whereas the 12 remaining -SH groups are located inside the mole and take part in the activity and the enzyme-substrate bond. It is concluded that Proteus TPases are molecules which have inverted quaternary structure in comparison to those of the coli group. The studied TPases have four subunits, each of them is constituted of one polypeptidic chain having a molecular weight of 55,000.

Escherichia↗
PubMed1975

[The effect of pyridoxal phosphate on the tryptophanases of five species of Enterobacteriaceae].

Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris et P. morganii tryptophanases (TPases) were studied for the spectral forms of the enzyme. The pH effect on the absorption spectrum and on the enzyme specific activity revealed that the coli group TPases are identical with but differ from Proteus TPases which differ themselves. The coli group TPases attach 4 mol of pyridoxal phosphate (PLP)/mol of enzyme, independently of the pH in the presence of K(plus) ions, and 9 mol of PLP/mol of enzyme must be reduced to achieve complete inactivation. The Proteus TPases attach 4 mol of PLP/mol of enzyme at PH 6.8, and 3 mol of PLP/mol of enzyme at pH 7.8 in K(plus) buffer. In P. morganii, 7 mol of PLP/mol of enzyme must be reduced to inactivate the enzyme, whereas P. vulgaris TPase cannot be completely inactivated by this method. These five TPases attach only 3 mol of PLP/mol of enzyme in a Na(plus) buffer, independently of the pH.

Calcium↗
PubMed1977

Monovalent cation activation of tryptophanase.

The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods. Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3). It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton. On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E. (1967) J. Biol. Chem; 242, 2800-2809. The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active. When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+. Two moles of thallium (I) were bound per mole of subunit. The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.

Binding Sites↗
PubMed1985

Assignment of ozone-sensitive tryptophan residue in tryptophanase by a dual-monitoring high-performance liquid chromatography system.

Tryptophanase purified from Escherichia coli B/1t7-A is inactivated by mild ozonization following pseudo-first-order kinetics. Previous data from the authors suggest that one out of two tryptophan residues (Trp's) in the enzyme subunit is preferentially oxidized concomitant with the ozone inactivation and has a direct interaction with the coenzyme, pyridoxal phosphate [PLP (M. Tokushige, Y. Fukuda, and Y. Watanabe, 1979, Biochem. Biophys. Res. Commun. 86, 976-981)]. To determine which Trp is more susceptible to ozonization and interacts with PLP, the native and ozonized enzyme proteins were cleaved by trypsin and the two Trp-containing peptides were analyzed by reverse-phase HPLC equipped with a dual-monitoring system consisting of an uv and a fluorescence monitor connected in tandem for selective detection of Trp-containing peptides. This device facilitated rapid detection and quantitation of the Trp-containing peptides which decreased upon ozonization. The results showed that Trp preferentially oxidized upon ozonization and involved in the interaction with PLP was the one in peptide T-15 rather than that in T-23, which Kagamiyama et al. originally designated (H. Kagamiyama, H. Wada, H. Matsubara, and E. E. Snell, 1972, J. Biol. Chem. 247, 1576-1585).

Binding Sites↗
PubMed1976

Structural and functional interdependence of the protomers of Escherichia coli K 12 tryptophanase during binding of pyridoxal 5'-phosphate.

It has been shown previously that the binding of pyridoxal 5'-phosphate to Escherichia coli K 12 tryptophanase brings about an important conformational change of the protein. The way in which this structural change is transmitted from holoprotomers to apoprotomers is investigated here, using hybrid molecules (between apoprotomers and irreversibly saturated holoprotomers). It is shown that the binding of two or three coenzyme molecules per tetramer stabilizes the whole molecule against thermal inactivation and cold-induced dissociation. The change in conformation induced on an apoprotomer by the proximity of three holoprotomers is described, using three structural probes: the kinetics of binding of the cofactor and of 5'-phosphopyridoxyl-tryptophan (an analog of an intermediate in the catalytic reaction), and the reactivity of the essential cysteines. The kinetic anticooperatively in the binding of pyridoxal 5'-phosphate is confirmed, some of its parameters are determined, and its mechanism is interpreted in relation to the coupling between the protomers.

Apoenzymes↗
PubMed1976

The dissociated tryptophanase subunit is inactive.

The dissociation into dimers of apotryptophanase has been studied from two points of view: the nature of the interactions which govern the dimer-tetramer equilibrium and the effect of dissociation on the functional properties of the enzyme. It is shown that the order in which different anions are able to shift the dimer-tetramer equilibrium is that of the Hofmeister series, thus showing that the main contribution to the interaction between two dimers is of hydrophobic nature. It is also shown that, when dimeric apotryptophanase is incubated in the presence of its cofactor and substrate, the kinetics of appearance of active molecules is of second order in enzyme and is independent of the pyridoxal-P concentration; its rate constant has been determined (5-10(4) M-1 S-1). These results indicate that the reassociation of dimers into tetramers is the rate-limiting step in the appearance of enzymatic activity, and that the tryptophanase dimer is not functional.

Anions↗