1,721,204 research outputs found
Reaction specificity of native and nicked 3,4-dihydroxyphenylalanine decarboxylase
No full text3,4-Dihydroxyphenylalanine (Dopa) decarboxylase is a stereospecific pyridoxal 5'-phosphate (PLP)-dependent cu-decarboxylase that converts L-aromatic amino acids into their corresponding amines, We now report that reaction of the enzyme with D-5-hydroxytryptophan or D-Dopa results in a time-dependent inactivation and conversion of the PLP coenzyme to pyridoxamine 5'-phosphate and PLP-D-amino acid Pictet-Spengler adducts, which have been identified by high performance liquid chromatography. We also show that the reaction specificity of Dopa decarboxylase toward aromatic amines depends on the experimental conditions. Whereas oxidative deamination occurs under aerobic conditions (Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C, (1996) J. Biol. Chem. 271, 23954-23959; Bertoldi, M,, Dominici, P., Moore, P, S,, Maras, B., and Borri Voltattorni, C. (1998) Biochemistry 37, 6552-6561), half-transamination and Pictet-Spengler reactions take place under anaerobic conditions. Moreover, we examined the reaction specificity of nicked Dopa decarboxylase, obtained by selective tryptic cleavage of the native enzyme between Lys(334) and His(335). Although this enzymatic species does not exhibit either decarboxylase or oxidative deamination activities, it retains a large percentage of the native transaminase activity toward D-aromatic amino acids and displays a slow transaminase activity toward aromatic amines, These transamination reactions occur concomitantly with the formation of cyclic coenzyme-substrate adducts. Together with additional data, we thus suggest that native Dopa decarboxylase can exist as an equilibrium among "open," "half-open," and "closed" forms
Reaction of dopa decarboxylase with L-aromatic amino acids under aerobic and anaerobic conditions
No full textAnalysis of the reaction of dopa decarboxylase (DDC) with L-dopa reveals that loss of decarboxylase activity with time is observed at enzyme concentrations approximately equal to the binding constant, K-d, of the enzyme for pyridoxal 5'-phosphate (PLP). Instead, at enzyme concentrations higher than K-d the course of product formation proceeds linearly until complete consumption of the substrate. Evidence is provided that under both experimental conditions no pyridoxamine 5'-phosphate (PMP) is formed during the reaction and that dissociation of coenzyme occurs at low enzyme concentration, leading to the formation of a PLP-L-dopa Pictet-Spengler cyclic adduct. Taken together, these results indicate that decarboxylation-dependent transamination does not accompany the decarboxylation of L-dopa proposed previously [O'Leary and Baughn (1977) J. Biol. Chem. 252, 7168-7173]. Nevertheless, when the reaction of DDC with L-dopa is studied under anaerobic conditions at an enzyme concentration higher than K-d, we observe that (1) the enzyme is gradually inactivated and inactivation is associated with PMP formation and (2) the initial velocity of decarboxylation is approximately half of that in the presence of O-2. Similar behaviour is observed by comparing the reaction with L-5-hydroxytryptophan occurring in aerobiosis or in anaerobiosis. Therefore the reaction of DDC with L-aromatic amino acids seems to be under O-2 control. In contrast, the reactivity of the enzyme with D-aromatic amino acids does not change in the presence or absence of O-2. These and other results, together with previous results on the effect exerted by O-2 on reaction specificity of DDC towards aromatic amines [Bertoldi, Frigeri, Paci and Borri Voltattorni (1999) J. Biol. Chem. 274, 5514-5521], suggest a productive effect of O-2 on an intermediate complex of the reaction of the enzyme with L-aromatic amino acids or aromatic amines
Ornithine and glutamate decarboxylases catalyse an oxidative deamination of their alpha-methyl substrates
No full textOrnithine decarboxylase (ODC) from Lactobacillus 30a catalyses the cleavage of α-methylornithine into ammonia and 2-methyl-1-pyrroline; glutamate decarboxylase (GAD) from Escherichia coli catalyses the cleavage of α-methylglutamate into ammonia and laevulinic acid. In our analyses, 2-methyl-1-pyrroline and laevulinic acid were identified by HPLC and mass spectroscopic analysis, and ammonia was identified by means of glutamate dehydrogenase. Molecular oxygen was consumed during these reactions in a 1:2 molar ratio with respect to the products. The catalytic efficiencies (kcat/Km) of the reactions catalysed by ODC and GAD were determined as 12500 and 9163 M-1 ̇min-1 respectively. When the reactions were performed under anaerobic conditions, no ammonia, 2-methyl-1-pyrroline or laevulinic acid was produced to a significant extent. The formation of ammonia and O2 consumption (in a 1:2 molar ratio with respect to ammonia) were also detected during the reaction of ODC and GAD with putrescine and γ-aminobutyrate respectively. Taken together, these findings clearly indicate that ODC and GAD catalyse an oxidative deamination of their decarboxylation products, a reaction similar to that catalysed by dopa decarboxylase (DDC) with α-methyldopa [Bertoldi, Dominici, Moore, Maras and Borri Voltattorni (1998) Biochemistry 37, 6552-6561]. Furthermore, this reaction was accompanied by a decarboxylation-dependent transamination occurring for GAD, DDC and ODC with a frequency of approx. 0.24%, 1% and 9% respectively compared with that of oxidative deamination.Ornithine decarboxylase (ODC) from Lactobacillus 30a catalyses the cleavage of alpha-methylornithine into ammonia and 2-methyl-1-pyrroline; glutamate decarboxylase (GAD) from Escherichia coli catalyses the cleavage of alpha-methylglutamate into ammonia and laevulinic acid. In our analyses, 2-methyl-1-pyrroline and laevulinic acid were identified by HPLC and mass spectroscopic analysis, and ammonia was identified by means of glutamate dehydrogenase. Molecular oxygen was consumed during these reactions in a 1.2 molar ratio with respect to the products. The catalytic efficiencies (k(cat)/K-m) of the reactions catalysed by ODC and GAD were determined as 12500 and 9163 M-1.min(-1) respectively. When the reactions were performed under anaerobic conditions, no ammonia, 2-methyl-1-pyrroline or laevulinic acid was produced to a significant extent. The formation of ammonia and O-2 consumption (in a 1:2 molar ratio with respect to ammonia) were also detected during the reaction of ODC and GAD with putrescine and gamma-aminobutyrate respectively. Taken together, these findings clearly indicate that ODC and GAD catalyse an oxidative deamination of their decarboxylation products. a reaction similar to that catalysed by dopa decarboxylase (DDC) with alpha-methyldopa [Bertoldi, Dominici, Moore, Maras and Borri Voltattorni (1998) Biochemistry 37, 6552-6561]. Furthermore, this reaction was accompanied by a decarboxylation-dependent transamination occurring for GAD, DDC and ODC with a frequency of approx. 0.24%, 1% and 9% respectively compared with that of oxidative deamination
THE INFLUENCE OF LACTOBACILLUS-PLANTARUM CULTURE INOCULATION ON THE FATE OF STAPHYLOCOCCUS-AUREUS AND SALMONELLA-TYPHIMURIUM IN MONTASIO CHEESE
No full textA novel high performance discrete flux integrator for control algorithms of fast rotating ac machines
Full text linkThis paper deals with the digital implementation of a motor control algorithm based on a unified machine model, thus usable with every traditional electric machine type (induction, brushless with interior permanent magnets, surface permanent magnets or pure reluctance). Starting from the machine equations in matrix form in continuous time, the paper exposes their discrete time transformation, suitable for digital implementation. Since the solution of these equations requires integration, the virtual division of the calculation time in sub-intervals is proposed to make the calculations more accurate. Optimization of this solver enables faster runs and higher precision especially when high rotating speed requires fast calculation time. The proposed solver is presented at different implementation levels, and its speed and accuracy performance are compared with standard solvers
Green tea polyphenols: novel irreversible inhibitors of dopa decarboxylase
No full textThe green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG), and (-)-epigallocatechin (EGC) were found to be inhibitors of Dopa decarboxylase (DDC). EG;CG; and EGC inactivate the enzyme in both a time- and concentration-dependent manner and exhibit saturation of the rate of inactivation at high concentrations, with efficiency of inactivation values (k(inact)/K-i) of 868 and 1511 M-1 min(-1), respectively. In contrast, gallic acid behaves as a weak inhibitor of DDC. Protection against inactivation by EGCG; and EG;C was observed in the presence of the active site-directed inhibitor D-Dopa. Either EGCG or EGC induce changes in the absorbance and CD bands of the visible spectrum of enzyme-bound PLP. Taken together, these findings indicate the active site nature of the interaction of DDC with both polyphenols. On the basis of the properties of the EGCG-inactivated enzyme, it can be suggested that inactivation could be ascribed to a covalent modification of not yet identified residue(s) of the active site of DDC. (C) 2001 Academic Press
Mutagenicity of pesticides evaluated by means of gene conversion in Saccharomyces cerevisiae and in Aspergiullus nidulans.
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