1,720,967 research outputs found
Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine)
No full textDopa decarboxylase (DDC) catalyzes not only the decarboxylation of L-aromatic amino acids but also side reactions including half-transamination of D-aromatic amino acids and oxidative deamination of aromatic amines. The latter reaction produces, in equivalent amounts, an aromatic aldehyde or ketone (depending on the nature of the substrate), and ammonia, accompanied by O(2) consumption in a 1 : 2 molar ratio with respect to the products. The kinetic mechanism and the pH dependence of the kinetic parameters have been determined in order to obtain information on the chemical mechanism for this reaction toward 5-hydroxytryptamine (5-HT). The initial velocity studies indicate that 5-HT and O(2) bind to the enzyme sequentially, and that D-Dopa is a competitive inhibitor versus 5-HT and a noncompetitive inhibitor versus O(2). The results are consistent with a mechanism in which 5-HT binds to DDC before O(2). The pH dependency of log V for the oxidative deaminase reaction shows that the enzyme possesses a single ionizing group with a pK value of approximately 7.8 that must be unprotonated for catalysis. In addition to an ionizing residue with a pK value of 7.9 similar to that found in the V profile, the (V/K)(5-HT) profile exhibits a pK value of 9.8, identical to that of free substrate. This pK was therefore tentatively assigned to the alpha-amino group of 5-HT. No titratable ionizing residue was detected in the (V/K)(O2) profile, in the pH range examined. Surprisingly, at pH values lower than 7, where oxidative deamination does not occur to a significant extent, a half-transamination of 5-HT takes place. The rate constant of pyridoxamine 5'-phosphate formation increases below a single pK of approximately 6.7. This value mirrors the spectrophotometric pK(spec) of the shift 420-384 nm of the external aldimine between DDC and 5-HT. Nevertheless, the analysis of the reaction of DDC with 5-HT under anaerobic conditions indicates that only half-transamination occurs with a pH-independent rate constant over the pH range 6-8.5. A model accounting for these data is proposed that provides alternative pathways leading to oxidative deamination or half-transamination
Dimerization and folding processes of Treponema denticola cystalysin: the role of pyridoxal 5'-phosphate.
No full textCystalysin, the key virulence factor in the bacterium Treponema denticola responsible for periodontitis, is a homodimeric pyridoxal 5'-phosphate (PLP)-C-S lyase. The dimerization process and the urea-induced unfolding equilibrium of holocystalysin were compared with those of the apo form. The presence of PLP decreases approximately 4 times the monomer-dimer equilibrium dissociation constant. By using a variety of spectroscopic and analytical procedures, we demonstrated a difference in their unfolding profiles. Upon the monomerization of apocystalysin, occurring between 1 and 2 M urea, a self-associated equilibrium intermediate with a very high beta-sheet content is stabilized over the 2.5-4 M urea range, giving rise to a fully unfolded monomer at higher urea concentrations. On the other hand, highly destabilizing conditions, accompanied by the formation of a significant amount of insoluble aggregates, are required for PLP release and monomerization. Refolding studies, together with analysis of the dissociation/association process of cystalysin, shed light on how the protein concentration and the presence or absence of PLP under refolding conditions could affect the recovery of the active dimeric enzyme and the production of insoluble aggregates. When the protein is completely denatured, the best reactivation yield found was approximately 50% and 25% for holo and apocystalysin, respectively. The dimerization and folding processes of cystalysin have been compared with those of another PLP C-S lyase, MalY from E. coli, and the possible relevance of their PLP binding mode in these processes has been discussed
Crystal structure of the apo form of human aromatic L-amino acid decarboxylase: an open conformation unveils novel insights into the mechanism of PLP addition to the apoenzymes of group II decarboxylases
No full textBehavior of fluorinated analogs of L-(3,4-dihydroxyphenyl)alanine and L-threo-(3,4-dihydroxyphenyl)serine as substrates for Dopa decarboxylase
No full textWe have determined the kinetic parameters for Dopa decarboxylase (DDC) of three ring-fluorinated analogs of 3, 4-dihydr-oxyphenylalanine (Dopa). The rank order of catalytic e0ciency of decarboxylation (kcat/Km)is Dopa > 6-F-Dopa > 2-F-Dopa > 5-F-Dopa. This rank is consistent with previous in vivo and in vitro studies which indicate that of the fluorinated analogs 6-F-Dopa has pharmacokinetics that are most suited for positron emission tomographic (PET) evaluation of dopamine function. The effectiveness of PET as a diagnostic tool the convenient half-life of 18F (110 min) and the favorable pharmacokinetics of 6-[18F]FDOPA have combined to make this an extremely valuable reagent to study dopaminergic activity. The reactions of the related fluorinated DOPS analogs show that while 6-F-threo-3, 4-(dihydroxyphenyl)serine (DOPS) is decarboxylated at approximately the same rate as the non-fluorinated substrate 2-F-threo-DOPS is not converted into the corresponding amine. In both cases a Pictet-Spengler condensation with the pyridoxal 5′-phosphate (PLP) cofactor occurs to produce tetrahydroisoquinolines. Condensation of fluorinated catecholamines and catechol amino acids with endogenous aldehydes will be investigated as an approach to study possible mechanisms of L-Dopa-linked neurotoxicity. Published by Elsevier Science (USA)
Limited tryptic proteolysis of pig kidney 3,4-dihydroxyphenylalanine decarboxylase
No full textPig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase can be nicked by trypsin with complete loss of its catalytic activity. The original dimer of subunit molecular weight of about 52,000 yields fragments of Mr 38,000 and 14,000, as seen on sodium dodecyl sulfate-gel electrophoresis. Though inactive, the nicked protein retains its native molecular weight and its capacity to bind pyridoxal-5'-phosphate (pyridoxal-P), is recognized by an antiserum raised against the native enzyme, and forms Schiff's base intermediates with aromatic amino acids in L and D forms. Thus, the nicked protein appears to be in a conformation--closely resembling that of the original enzyme--which consists of a tight association of the two tryptic fragments. Dissociation and separation of the two fragments can be achieved under denaturing conditions on a reverse-phase HPLC column. The pyridoxal-P binding site is located on the larger fragment. No NH2-terminal residue is detected in either the intact enzyme or the larger fragment, whereas analysis of the smaller fragment yields a sequence of the first 50 amino acid residues. These data indicate that the smaller fragment is located at about one-third from the COOH terminus of Dopa decarboxylase, while the larger fragment constitutes the aminic portion of the molecule. The site of trypsin cleavage seems to be in a region of the enzyme particularly susceptible to proteolysis. The results of these studies contribute to a better understanding of the structural properties of pig kidney Dopa decarboxylase and may constitute an important step toward the elucidation of the enzyme's primary structure
S81 L and G170R mutations causing Primary Hyperoxaluria Type I in homozygosis and heterozygosis: an example of positive interallelic complementation.
No full textPrimary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5'-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients
S81 L and G170R mutations causing Primary Hyperoxaluria Type I in homozygosis and heterozygosis: an example of positive interallelic complementation.
No full textPrimary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5'-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients
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