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Multiple roles of the active site lysine of Dopa decarboxylase
No full textThe pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of L-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of L-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation. (C) 2009 Elsevier Inc. All rights reserved
The steady state kinetics of tyrosine decarboxylase from Streptococcus faecalis
No full textThe present study has explained the general reaction mechanism of the bacterial tyrosine decarboxylase. The rate equation for this mechanism has been presented. The steady state kinetics of tyrosine decarboxylase, as for tyrosine transaminase, have shown that the apoenzyme can bind not only the coenzyme, but also the non-enzymatically formed Schiff base between the coenzyme and the substrate. Our data then have confirmed the importance of the non-enzymatically formed Schiff base in the B6-dependent enzymes, possibly in all of them which have a low affinity constant for the coenzyme, such that the coenzyme must be present in excess in respect to the protein to saturate the active center. The interaction between apotyrosine decarboxylase with pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate has been studied
Dopa decarboxylase
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Reaction and substrate specificity of pig kidney Dopa decarboxylase under aerobic and anaerobic conditions
No full textDopa decarboxylase (DDC) catalyzes as main reaction the stereospecific CO2 abstraction from L-Dopa and L-5-hydroxytryptophan (5-HTP), generating the corresponding aromatic amines, dopamine and serotonin, respectively. Side reactions with turnover time of minutes are also catalyzed by the enzyme. In particular, DDC exhibits half-transaminase activity toward D-aromatic amino acids and oxidative deaminase activity toward aromatic amines. The latter reaction could represent a new activity for this class of enzymes. Studies on the effect exerted by O2 on reaction specificity of DDC revealed that under anaerobic conditions decarboxylation of L-aromatic amino acids takes place with a kcat approximately half of that measured in the presence of O2, and is accompanied by a decarboxylation- dependent transamination, whereas oxidative deamination of aromatic amines is replaced by half-transamination. Half-transamination of D-aromatic amino acids is unaffected by the presence or absence of O2. Some structural elements relevant for the control of reaction and substrate specificity of DDC have been identified by means of limited tryptic digestion and site-directed mutagenesis studies. All together, the data indicate that the chemical nature of the substrate, the presence of O2, the integrity of a mobile loop, the absence of perturbation in the coenzyme-binding cleft and pH are important requirements for the achievement of a closed conformational state where the highest level of reaction specificity is reached. © 2003 Elsevier Science B.V. All rights reserved
Alpha5 pyridoxalacetic acid and alpha5 pyridoxyl-L-phenylalanine acetic acid: their action on some B6-dependent enzymes
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The binding of nucleotides to 3'-nucleotidase from wheat germ
No full textThe 3'-mononucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) from wheat germ has been purified 2,000 fold. The enzyme has a molecular weight of approximatley 32,000, as judged by the use of G-100 gel filtration, and does not attack 2'- or 5'- nucleotides. In order to obtain some indications on the structural requirements for binding and reactivity, the purified enzyme has been subjected to kinetic analyses, including initial velocities with several 3'-ribomononucleotides, inhibition by 5'- nucleotides and nucleotide-analogues, and effect of pH and sulphydryl compounds. The data indicate one base binding site at the active site of the enzyme. This site appears to be the same involved in the binding of both substrates and inhibitors, with higher affinity for purine nucleotides than for pyrimidine compounds, in the order guanosine greater than adenosine greater than inosine greater than uridine greater than cytidine nucleotides
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