Studies on the metabolism of tryptophan and antharnilic acid in plants.

Abstract

Part I. Tryptophan biosynthesis in Bengal gram In the present investigation, tryptophan synthetase has been detected in cell?free extracts of Bengal gram (Cicer arietinum). This enzyme catalyses the condensation of indole and L?serine with the formation of tryptophan. The amount of tryptophan formed in the reaction was estimated quantitatively and the stoichiometry of the reaction established. The Bengal gram enzyme showed a definite requirement for pyridoxal phosphate. With the crude enzyme extract the optimum pH of the reaction was 5.5 and the Michaelis–Menten constant with respect to indole at a constant serine concentration of 10?² M was 0.57 × 10?³ M. Heavy metal ions like Hg and Ag were inhibitory. Carbonyl reagents like hydroxylamine and isonicotinic acid hydrazide also inhibited the reaction. This indicated that tryptophan synthetase was a pyridoxal phosphate enzyme. It was found that 4?nitrosalicylaldehyde could substitute for pyridoxal phosphate in the reaction. A study of the effect of enzyme concentration (crude Bengal gram extract) showed a diminution of enzyme activity with increasing amounts of enzyme preparation. This suggested the presence of a natural inhibitor in the crude extracts of Bengal gram. It was also observed that tryptophan synthetase activity of the seed extracts could be enhanced by dilution. Dialysis against water was not effective in removing the inhibition. However, by dialysing the enzyme extract against EDTA, the inhibition was removed. This indicated that a metal ion was probably involved. Further studies showed that the inhibitor was stable to boiling or even ashing. The ash after dissolving in dilute hydrochloric acid gave a positive reaction for ferric ion when tested with potassium ferrocyanide. Cyanide (4 × 10?³ M) and o?phenanthroline reversed the inhibition, while 8?hydroxyquinoline was ineffective. These findings suggested that the natural inhibitor in Bengal gram was ferrous iron. After these exploratory studies, purification of the enzyme was undertaken. A 220?fold purification of the enzyme was achieved by MnSO? treatment, negative adsorption on alumina C?gel, adsorption on tricalcium phosphate and elution with phosphate buffer (pH 7.2) containing ammonium sulphate and serine. The general properties of the purified enzyme, with special reference to the effect of substrate concentration, time?course of reaction, optimum pH, effect of temperature, action of inhibitors and cofactor requirements were studied and compared with the properties of the bacterial and fungal enzymes. The inhibition caused by isonicotinic acid hydrazide and L?penicillamine, the well?known inhibitors of enzymes requiring pyridoxal phosphate, were studied in detail. The inhibition caused by these reagents was completely reversed by either pyridoxal phosphate or nitrosalicylaldehyde, and to a lesser extent by pyridoxal. Indolepropionic acid, a structural analogue of indole, was found to be a potent inhibitor for this reaction. A detailed study of this inhibition showed that it was of the competitive type. Systematic studies carried out on the kinetics of the enzyme reaction using the purified enzyme showed that the Km value for indole was 0.66 × 10?³ M at a constant serine concentration of 8 × 10?? M and the Km values for L? and DL?serine at a constant indole concentration of 7 × 10?³ M were 4.1 × 10?³ M and 8.06 × 10?³ M respectively.

Part II. Anthranilic acid oxidation in Tecoma stans Anthranilic acid, an important intermediate of tryptophan metabolism, was found to be oxidised to catechol by an enzyme system obtained from the leaves of Tecoma stans. In this connection a highly sensitive colorimetric method for the estimation of catechol was developed. The properties of the anthranilic acid oxidase system, which was found to reside in the chloroplast fraction of Tecoma leaves, were studied. The optimum pH of the reaction was 5.0. Anthranilic acid at concentrations higher than 10?³ M was highly inhibitory to the chloroplast enzyme. The enzyme was inhibited by atebrine and p?chloromercuribenzoate. The enzyme was highly specific for anthranilic acid. Under the experimental conditions employed, benzoic, salicylic and pyrocatechuic acids were not attacked. The stoichiometry of the reaction was established by estimating the amount of anthranilic acid disappearing and the amounts of catechol and ammonia formed. The enzyme was obtained in a cell?free condition and further purified by isoelectric precipitation and ammonium sulphate fractionation. The general properties of the purified enzyme preparation were studied in detail. In the case of the purified enzyme, the optimum substrate concentration was 10?? M and there was no abrupt fall in activity with increase in the concentration of anthranilic acid, as was observed in the case of the chloroplast enzyme. Studies on the action of various inhibitors indicated the participation of a multi?enzyme sequence in the overall reaction. TPNH and tetrahydrofolic acid activated the enzyme system, thus underlining the similarity of mechanisms of this reaction and the phenylalanine hydroxylase system present in mammalian liver.