An essential arginine residue at the binding site of pig kidney 3,4-dihydroxyphenylalanine decarboxylase

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by the arginine-specific reagent phenylglyoxal. Under these experimental conditions, the reaction follows pseudo-first-order kinetics with a second-order rate constant of 25 m-1 min-1. Holo- and apo-enzyme were inactivated at the same rate. However, inactivation seems to be related to modification of 1 and 2 arginyl residues per mol of holo- and apo-enzyme, respectively. Only one of these two residues was essential to decarboxylase activity of the enzyme. Phenylglyoxal-modified apo-Dopa decarboxylase retained the capacity to bind pyridoxal-P. Neither this reconstituted species nor the phenylglyoxal-modified holoenzyme were able to form Schiff base intermediates with aromatic amino acids in L and D forms. These data together with protection experiments suggest that the susceptible arginine residue in holoenzyme may somehow perturb the substrate binding site. However, unlike in other pyridoxal-P enzymes, this critical arginine in Dopa decarboxylase does not seem to behave as an anionic recognition site for the phosphate group of the coenzyme or the carboxy group of the substrate. It is speculated that this guanidyl group could function in hydrogen bonding of substrate side chain.     

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.     

Chemical modification of pig kidney 3,4-dihydroxyphenylalanine decarboxylase with diethyl pyrocarbonate. Evidence for an essential histidyl residue

Diethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme.     

Purification and characterization of 3,4-dihydroxyphenylalanine decarboxyase from pig kidney.

A procedure for 3,4-dihydroxyphenylalanine decarboxylase from pig kkdney purification is described in detail. The preparation has no detectable impurity on electrophoresis and on ultracentrifugation and authors. However two significant differences are observed: a different stimulation of activity by added pyridoxal 5'-phosphate and a nearly complete decarboxylation of L-3,4-dihydroxyphenylalanine in absence of added coenzyme. Absorption, fluorescence and circular dichroism properties of the coenzyme-apoenzyme interaction are also described. The results are consistent with the existence of at least four coenzyme-apoenzyme complexes, three of them active.     

Purification and characterization of rat-liver 3,4-dihydroxyphenylalanine decarboxylase

A simple and rapid procedure, which takes advantage of the effectiveness of conventional and HPLC hydrophobic interaction, for the isolation of highly purified rat liver 3,4-dihydroxyphenylalanine decarboxylase is described in detail. Some of its structural and functional properties are reported and discussed in comparison with those of pig kidney 3,4-dihydroxyphenylalanine decarboxylase.     

Decarboxylation-dependent transamination catalyzed by mammalian 3,4-dihydroxyphenylalanine decarboxylase

In addition to the usual decarboxylation, pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase catalyzes a decarboxylation-dependent transamination which converts dopa into 3,4-dihydroxyphenylacetaldehyde and sinultaneously converts enzyme-bound pyridoxal-P into pyridoxamine-P. Similar reactions occur when this enzyme acts on m-tyrosine, alpha-methyldopa, and alpha-methyl-m-tyrosine. The transamination occurs in about 0.02% of decarboxylations of dopa and m-tyrosine and in about 2% of decarboxylations of alpha-methyldopa and alpha-methyl-m-tyrosine. The fraction of decarboxylations proceeding by the transamination pathway is independent of pH. This reaction appears to result from a divergence in the normal mechanism of decarboxylation; the quinoid intermediate which is formed by decarboxylation of the substrate-pyridoxal-P-Schiff base ordinarily protonates on the alpha carbon of the amino acid, but protonation occasionally occurs at the benzylic carbon of the coenzyme, and this latter route leads to transamination.     

Reaction specificity of native and nicked 3,4-dihydroxyphenylalanine decarboxylase

3,4-Dihydroxyphenylalanine (Dopa) decarboxylase is a stereospecific pyridoxal 5'-phosphate (PLP)-dependent alpha-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 Lys334 and His335. 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.     

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.     

Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.     

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO₂, NH_3, and H₂O₂. This contrasts to the typical DDC-catalyzed reaction, which produces CO₂ and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H₂O₂ in the process. Biochemical assessment established that H₂O₂, formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H₂O; thereby avoiding oxidative stress by H₂O₂. Results of our structural and functional analyses provide a reasonable explanation of mechanisms involved in DHPA synthase-mediated reactions. Based on the key active site residue Asn192, identified in Drosophila DHPA synthase, we were able to distinguish all available insect DHPA synthases from DDC sequences primarily.     

Dye-sensitized photo-oxidation of pig kidney Dopa decarboxylase

The effects of irradiation of pig kidney Dopa decarboxylase by visible light absorbed by the intrinsic chromophore, pyridoxal-P, and by the externally added dyes, pyridoxal-P or proflavin, have been studied. In all cases inactivation was observed, even though to different extens, which seemed to be essentially correlated to tryptophanyl residues photodestruction. Kinetics of inactivation and oxidation of these amino acid residues revealed the presence of two distinct groups of tryptophan residues with different photooxidation rate constants. A different role for these classes of residues in the structure and function of Dopa decarboxylase has been suggested.     

Limited proteolysis of pig liver CoA synthase: evidence for subunit identity

The bifunctional enzyme CoA synthase can be nicked by trypsin without loss of its activities. The original dimer of subunit Mr approx. 61 000 yields fragments of Mr 41 000 and 22 000 as seen on gel electrophoresis in the presence of SDS, but the nicked enzyme retains the native Mr of 118 000. Further proteolysis occurs rapidly in the absence of protecting substrates. The N-terminal of native CoA synthase is proline, and proteolysis exposes glycine as a second N-terminal. This evidence strongly suggests that the subunits are identical.     

Oxygraphic assay of 3,4-dihydroxyphenylalanine decarboxylase activity by coupled reaction with free and immobilized serum amine oxidase

An oxygraphic method for the assay of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase activity which makes use of the coupled reactions of Dopa decarboxylase with serum amine oxidase is presented. Both free and immobilized amine oxidases were utilized. The assay is simple, rapid, and allows a continuous monitoring of the reaction. The kinetic parameters for Dopa decarboxylase obtained with the coupled assay do not significantly differ from those obtained by standard methods.     

Radical sequestration by protein-bound 3,4-dihydroxyphenylalanine

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA), a redox-active product of protein oxidation, is capable of functioning as both a pro- and antioxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Being the major treatment available for Parkinson's disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. We hypothesise that the generation of PB-DOPA can trigger an enhancement of the cellular antioxidant defence system, thus enabling PB-DOPA to restrict and potentially terminate the initiating oxidative stress, minimising the level of oxidative damage. Using luminol-enhanced chemiluminescence, we demonstrate that free DOPA is capable of direct peroxyl radical scavenging, even in the presence of competing scavengers, and has a different effect to that of the parent amino acid, tyrosine. Furthermore, we show that both free and PB-DOPA, in combination or individually, were able to protect monocytes and macrophages from peroxyl radical-induced oxidative stress in vitro. These results confirm a role for both free and PB-DOPA in cellular antioxidant defences and suggest the possibility of using DOPA as a potential therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage.     

Infrared-assisted tryptic proteolysis for peptide mapping

In this report, infrared (IR) radiation was employed to enhance the efficiency of tryptic proteolysis for peptide mapping. Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of BSA and myoglobin (MYO) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 69% (BSA) and 90% (MYO) that were much better than those obtained by conventional in-solution tryptic digestion. The present IR-assisted proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.     

Stereospecificity of sodium borohydride reduction of pig kidney dopa decarboxylase

Sodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced epsilon-[3H]pyridoxyllysine. Degradation of this material to 4'-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4' of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external Schiff's base, attempts to trap reaction intermediates were made by reduction with sodium boro [3H]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as epsilon-N-pyridoxyllysine. These results suggest that the internal Schiff's base is in mobile equilibrium with the external Schiff's base and that sodium borohydride reduction displaces this equilibrium, resulting in complete reduction of the internal Schiff's base.