Reversible unfolding of the β2 subunit of Escherichia coli tryptophan synthetase and its proteolytic fragments

The guanidine hydrochloride-induced reversible unfolding transitions at 4 °C of the β₂ subunit of tryptophan synthetase (l-serine hydrolyase (adding indole) EC. 4.2.1.20) and of its two proteolytic fragments, F₁ and F₂, are compared. The unfolding of the β₂ subunit shows a multistate behaviour, as judged by circular dichroism and fluorescence measurements. When isolated, the two fragments have different stabilities. Within β₂, the region corresponding to the large fragment, F₁ behaves as the corresponding isolated fragment, and no stabilization arising from the interaction with the complementary fragment can be detected. The same behaviour is suggested for the small fragment, F₂. These results lead to the apparent conclusion that, at least under these experimental conditions, the interactions between domains do not contribute greatly to the energetics of the folding process of the large β₂ protein.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate

The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit.Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.     

Evidence for the involvement of a rapidly turning over protease in the degradation of cytochrome oxidase in Neurospora crassa

The reaction of L-aromatic aminoacid decarboxylase (EC 4.1.1.28) with α-methyl-L-DOPA or 5-hydroxy-L-tryptophan leads to the formation of dihydroxyphenylacetone or, respectively, 5-hydroxyindolacetaldeyde. These are produced in amounts far exceeding, on molar basis, that of the coenzyme, pyridoxal-5′-phosphate. The reaction cannot therefore be simply a decarboxylation-dependent transamination, using the coenzyme as an amino group acceptor. Evidence is presented which rules out the possibility that this phenomenon is due to an oxidative deamination.     

Spatial structure and the mechanism of tyrosine phenol-lyase and tryptophan indole-lyase

Bacterial tyrosine phenol-lyase [EC 4.1.99.2] and tryptophan indole-lyase [EC 4.1.99.1] are pyridoxal 5′-phosphate dependent β-eliminating lyases that catalyze the reversible decomposition of L-tyrosine and L-tryptophan to pyruvate, ammonia, and phenol or indole, respectively. This review considers the three-dimensional structures of the holoenzymes of tyrosine phenol-lyase and tryptophan indole-lyase and several enzyme-inhibitor complexes that model distinct reaction stages of the β-elimination of L-tyrosine. The structural basis of the influence of monovalent cations on enzymatic activity is discussed. Studies of the spectral and catalytic properties of mutant enzymes made it possible to elucidate the catalytic functions of a number of amino acid residues and to conclude that the acid-base properties of the catalytic groups are optimal for catalysis in the hydrophobic active sites of tyrosine phenol-lyase and tryptophan indol-lyase and differ from those in water solutions. A study of the mechanisms of labilization of the Cα proton of the bound amino acids and activation of the leaving groups of the substrates during the catalytic process demonstrated that the reaction proceeds via concerted, rather than stepwise, pathways in certain cases.