Reactions of DOPA (3,4-dihydroxyphenylalanine) decarboxylase with DOPA.

The study of DOPA (3,4-dihydroxyphenylalanine) decarboxylase by steady-state methods is difficult because multiple reactions occur. The reaction with DOPA was studied at enzyme concentrations between 20 and 50 micrometer by direct observation of the bound coenzyme by using stopped-flow and conventional spectrophotometry. Four processes were observed on different time scales and three of these were attributed to stages in the decarboxylation. The fourth was attributed to an accompanying transamination that renders the enzyme inactive. It was clear that much, if not all, of the 330 nm-absorbing coenzyme present in the free enzyme plays an active part in the decarboxylation, since it is converted into 420 nm-absorbing material in the first observable step. An intermediate absorbing maximally at 390 nm is formed in a slower step. Rate and equilibrium constants have been determined and the ratio of decarboxylation to transamination was estimated to be 1200:1.     

Neuronal metabolism and DOPA decarboxylase immunoreactivity in terminal noradrenergic sympathetic axons of rat.

This study was undertaken to determine whether immuno-histochemical staining for DOPA decarboxylase (DDC) is present in axons of rat noradrenergic sympathetic neurons. A sparse plexus of varicose axons exhibiting DDC-like immunoreactivity (DDC-IR) was associated with blood vessels and acini in the submandibular gland, but this was much less extensive than the population that exhibited tyrosine hydroxylase-like immunoreactivity (TH-IR). The varicose terminal TH-IR axons in atrium, spleen, and vas deferens were devoid of DDC-IR both in grown rats and during the post-natal period of axon growth, although weak DDC-IR was seen in large pre-terminal nerve bundles. Similar patterns of staining were seen with paraffin-embedded and with frozen, formaldehyde-fixed material. No enhancement of DDC-IR was seen in any tissue after chronic alteration of catecholamine turnover with reserpine or alpha-methyl-para-tyrosine, and the numbers of submandibular DDC-IR axons were not increased by disruption of axonal transport with colchicine or by decentralization of the superior cervical ganglion. We conclude that terminal noradrenergic axons contain insufficient DDC-IR for microscopic visualization, regardless of their metabolic state, reinforcing previous evidence that DDC-IR can be used as a histochemical marker for dopaminergic axons. By this criterion, the rat submandibular gland may receive a sparse dopaminergic innervation.     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.     

Characterization of highly purified ornithine decarboxylase from rat heart

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5- nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.     

The chymotryptic phosphopyridoxyl peptide of DOPA decarboxylase from pig kidney

The amino acid sequence of the coenzyme-binding site of DOPA-decarboxylase from pig kidney has been determined. A sample of enzyme was reduced with NABH₄, aminoethylated and then digested with chymotrypsin. A single phosphopyridoxyl peptide was isolated and its sequence proved to be: Asn-Phe-Asn-Pro-His-Lys(Pxy)-Trp. Sequence homologies at the active site of various pyridoxalphosphate enzymes, and particularly of the bacterial decarboxylase, are discussed, with some emphasis on the constant presence of a histidine residue adjacent to the phosphopyridoxyl-lysine.     

Insights into the mechanism of oxidative deamination catalyzed by DOPA decarboxylase

The unusual oxygen-consuming oxidative deamination reaction catalyzed by the pyridoxal 5'-phosphate (PLP) enzyme DOPA decarboxylase (DDC) was here investigated. Either wild-type or Y332F DDC variant is able to perform such oxidation toward aromatic amines or aromatic l-amino acids, respectively, without the aid of any cofactor related to oxygen chemistry. Oxidative deamination produces, in equivalent amounts, a carbonyl compound and ammonia, accompanied by dioxygen consumption in a 1:2 molar ratio with respect to the products. Kinetic studies either in the pre-steady or in the steady state, together with HPLC analyses of reaction mixtures under varying experimental conditions, revealed that a ketimine accumulates during the linear phase of product formation. This species is reactive since it is converted back to PLP when the substrate is consumed. Rapid-mixing chemical quench studies provide evidence that the ketimine is indeed an intermediate formed during the first catalytic cycle. Moreover, superoxide anion and hydrogen peroxide are both generated during the catalytic cycles. On this basis, a mechanism of oxidative deamination consistent with the present data is proposed. Furthermore, the catalytic properties of the T246A DDC mutant together with those previously obtained with H192Q mutant allow us to propose that the Thr246-His192 dyad could act as a general base in promoting the first step of the oxidative deamination of aromatic amines.     

Crystallization and preliminary X-ray analysis of pig kidney DOPA decarboxylase.

DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.     

Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors

Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 μM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.     

Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a highly inducible protein whose expression involves a complex and variable array of regulatory mechanisms. We investigated the influence of the 5'-untranslated region (5'UTR) of the rat ODC mRNA on translation of the mRNA in a cell-free system and in cultured mammalian cells. ODC mRNA containing the full-length 5'UTR was translated in reticulocyte lysates at approximately 5% of the rate of mRNA containing no ODC 5' leader sequences. The complete 5'UTR inhibited expression of a heterologous gene product, human growth hormone, to the same extent in cultured mammalian cells. Furthermore, the 5'-most 130 bases of the rat ODC 5'UTR, a conserved G/C-rich region predicted to form a stable stem-loop structure (delta G = -68 kcal/mol), repressed translation to the same extent as the entire 5'UTR, both in the lysates and in intact cells. The 3'-most 160 bases of the 5'UTR, containing a small upstream open reading frame, decreased expression by 50-65% both in vitro and in intact cells, compared with controls lacking any ODC 5'UTR sequences. Mutation of the initiation codon AUG beginning this upstream open reading frame to GCG restored expression to rates equivalent to those seen in constructions containing no ODC 5'UTR sequences. We conclude that the rat ODC mRNA 5'UTR can inhibit translation of ODC mRNA both in vitro and in vivo, and that the predicted stem-loop structure at the 5' end of the 5'UTR is both necessary and sufficient for this inhibition.     

Long-term increase of glutamate decarboxylase mRNA in a rat model of temporal lobe epilepsy

Behavioral changes following injury, neural degeneration, and aging partly reflect the synaptic plasticity of the nervous system. Such long-term plastic changes are likely to depend on alterations in the production of proteins involved in synaptic structures and neurotransmission. We have studied the regulation of the mRNA encoding one such protein, glutamate decarboxylase (GAD), the rate limiting enzyme of GABA synthesis, after a unilateral lesion in the hippocampus that leads to increased seizure susceptibility. Quantitative in situ hybridization reveals a long-term increase in GAD mRNA in several bilateral structures, as well as in specific neurons in the ipsilateral dentate gyrus. Our data do not support the often stated hypothesis that seizure susceptibility depends on the malfunction of GABA neurons.