Kinetic and equilibrium studies on the activation of Escherichia coli K12 tryptophanase by pyridoxal 5'-phosphate and monovalent cations. (41/1268)

An improved purification of Escherichia coli K12 tryptophanase is presented. It is shown that the apoenzyme crystals, oxidized by exposure to air, can be reactivated by treatment with a reducing agent. The titration of sulfhydryl groups shows that four --SH groups are exposed and two are masked per protomer. The influence of two effectors, monovalent cations and the coenzyme pyridoxal 5'-phosphate, on the reactivity of --SH groups and the enzymatic activity was investigated. The --SH groups react more slowly in holo- than in apoenzyme in the presence of potassium ions. If these ions are replaced by sodium ions, the reactivity becomes the same. Potassium and ammonium ions, both activators, give sigmoidal activation curves. The sodium ion is a Michaelian inhibitor of potassium activation. The binding of pyridoxal 5'-phosphate was examined by kinetics and at equilibrium. The kinetics are shown to be very slow; the rate constants of the forward and reverse reactions have been measured. The binding equilibrium, examined with 3H-labeled pyridoxal 5'-phosphate, gives one site per protomer with a K-D value of (3.2 plus or minus 0.8) times 10-7 M. The K-m for pyridoxal-P was determined by activity measurements. The binding equilibrium is attained after several hours, giving a value of 4.2 times 10-7 M, being nearly identical with the dissociation constant and 5 times smaller than previously reported.  (+info)

Expression of prokaryotic and eukaryotic cytochromes c in Escherichia coli. (42/1268)

C-type cytochromes from various sources show substantial structural conservation. For the covalent attachment of heme groups to apocytochromes, however, three different enzyme systems have been described so far. We have examined the ability of the heme ligation systems of Escherichia coli and of Saccharomyces cerevisiae to process cytochromes from S. cerevisiae, Paracoccus denitrificans, and Synechocystis sp. PCC 6803. E. coli's maturation system with at least eight different proteins accepted all these cytochromes for heme ligation. The single subunit heme lyase from S. cerevisiae mitochondria, on the other hand, failed to attach heme groups to cytochromes of prokaryotic origin.  (+info)

Existence of two levels of repression in the biosynthesis of methionine in Saccharomyces cerevisiae: effect of lomofungin on enzyme synthesis. (43/1268)

Derepression of a methionine biosynthetic enzyme (homocysteine synthase) has been studied after repression either by exogenous methionine or by exogenous S-adenosylmethionine (SAM). Lomofungin, which inhibits the synthesis of ribosomal precursor and messenger ribonucleic acid but not of protein in Saccharomyces cerevisiae, has been used in this system. It has been shown that the addition of this antibiotic prevents the derepression of homocysteine synthase after repression by exogenous methionine but not after repression by exogenous SAM. These experiments with lomofungin and the kinetics of repression after addition of methionine or SAM to the growth medium provide evidence that the repression induced by exogenous methionine acts at the transcriptional level whereas the repression induced by exogenous SAM acts at the translational level.  (+info)

Cysteine synthase (O-acetylserine (thiol) lyase) substrate specificities classify the mitochondrial isoform as a cyanoalanine synthase. (44/1268)

A cyanoalanine synthase and two isoforms (A, cytosolic and B, chloroplastic) of cysteine synthase (O:-acetylserine (thiol) lyase) were isolated from spinach. N-terminal amino acid sequence analysis of the cyanoalanine synthase gave 100% homology for the determined 12 residues with a published sequence for the mitochondrial cysteine synthase isoform. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions, although with different efficiencies. Michaelis-Menten kinetics were observed for all three enzymes when substrate saturation experiments were performed varying O:-acetylserine, chloroalanine and cysteine. Negative co-operative kinetics were observed for cysteine synthases A and B when substrate saturation experiments were performed varying sulphide and cyanide, compared with the Michaelis-Menten kinetics observed for cyanoalanine synthase. The exception was negative co-operativity observed towards sulphide for cyanoalanine synthase with O:-acetylserine as co-substrate. The optimum sulphide concentration was dependent on the alanyl co-substrate used. The amino acid sequence similarity places these three enzymes in the same gene family, and whilst the close kinetic similarities support this, they also indicate distinct roles for the isoforms.  (+info)

The kinetic of degradation of chondroitin of sulphates and hyaluronic acid by chondroitinase form Proteus vulgaris. (45/1268)

Km and Vmax. were determined for the degradation by chondroitinase of chondroitin 4-sulphate, 4-sulphate-proteoglycna, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid. Degradation of chondroitin 4-sulphate was inhibited by hyaluronic acid but not by keratan sulphate. The results are discussed with regard to the use to the use of chondroitinase as a sleective reagent for the degradation of tissue glycosaminoglycans.  (+info)

Accumulation of arginine precursors in Escherichia coli: effects on growth, enzyme repression, and application to the forward selection of arginine auxotrophs. (46/1268)

The accumulation or ornithine, citrulline, and possibly acetylornithine by Escherichia coli K-12 arginineless mutants provided with acetylarginine as source of arginine causes severe growth inhibition. This occurs under conditions where comparable derivatives of E. coli W (Bollon and Vogel, 1973) show little or no growth inhibition. The same conditions, which have been reported to cause noncorrelative synthesis of acetylornithinase and argininosuccinase in E. coli W (Bollon and Vogel, 1973), do not alter the correlative pattern of enzyme synthesis observed in E. coli K-12. Moreover, previously reported effects of ornithine and citrulline on repression of the arginine regulon in E. coli W are not observed in the K-12 strains examined. The bearing of these observations on possible differences between the mechanism of enzyme repression operating in the two types of strains cannot yet be fully evaluated; it is, however, clear that considerable care should be exercised before extrapolating the results obtained with one type of strain to the other one. The particularly strong inhibition of acetylarginine utilization exerted by ornithine in E. coli K-12 allows the forward selection of several classes of arginine auxotrophs from strains deficient in carbamoylphosphate biosynthesis and thus capable of ornithine accumulation. Possible applications of this technique to the genetic analysis of the bipolar argECBH operon are discussed.  (+info)

Studies on the mechanism of inhibition of Salmonella typhimurium by 1,2,4-triazole. (47/1268)

The inhibition of Salmonella typhimurium by 1,2,4-triazole appears to be mediated through an effect on L-cysteine biosynthesis. O-Acetylserine sulfhydrylase A, the final enzyme in the L-cysteine biosynthetic pathway, was found to catalyze a reaction (triazolylase) between O-acetyl-L-serine and 1,2,4-triazole, giving 1,2,4-triazole-1-alanine as a product. In wild type S. typhimurium grown on 4 mM 1,2,4-triazole, 97% of the total O-acetyl-L-serine synthesized in vivo is incorporated into 1,2,4-triazole-1-alanine. 1,2,4-triazole also significantly lowers the levels of several of the enzymes necessary for sulfate reduction. This effect is presumably due to the ability of the inhibitor to lower intracellular concentrations of O-acetyl-L-serine, an inducer of these enzymes. Inhibition of growth is probably caused by L-cysteine starvation, arising from the decreased availability of the L-cysteine precursors, sulfide and O-acetyl-L-serine. Two 1,2,4-triazole-resistant strains bearing mutations in cysK, the structural gene for O-acetylserine sulfhydrylase A, incorporate only small quantities of O-acetyl-L-serine into 1,2,4-triazole-1-alanine in vivo. In vitro studies, using purified preparations of O-acetylserine sulfhydrylase A, revealed greater losses of triazolylase activity than sulfhydrylase activity in the enzymes from both cysK mutants. Resistance to 1,2,4-triazole apparently can arise from mutations leading to a preferential loss of triazolylase activity or from mutations which diminish both activities to the extent that high concentrations of O-acetyl-L-serine and sulfide accumulate behind the sulfhydrylase reaction.  (+info)

A glyoxalase I inhibitor of a new structural type produced by Streptomyces. (48/1268)

Many streptomyces strains produced an inhibitor of crude glyoxalase prepared from rat liver which did not inhibit glyoxalase I prepared from yeast. Another inhibitor, C11H14O6, which inhibited glyoxalases prepared from both rat liver and yeast was obtained from a cultured broth of Streptomyces griseosproeus and crystallized. Preincubation of this inhibitor with reuduced glutathione increased its inhibitory activity, which suggested its reaction with reduced glutathione. It showed a strong inhibition of growth of HeLa cells and inhibition of Ehrlich ascites carcinoma by daily injection. It also showed weak inhibition of the solid type of Ehrlich carcinoma and prolonged the survival period of mice inoculated with L-1210 cells.  (+info)