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.     

Green tea polyphenols: novel irreversible inhibitors of dopa decarboxylase

The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG), and (-)-epigallocatechin (EGC) were found to be inhibitors of Dopa decarboxylase (DDC). EGCG 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 EGC 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.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site.

The biodegradative ornithine decarboxylase of Escherichia coli has been purified to apparent homogeneity. At its pH optimum (pH 7.0), the enzyme exists as a dimer of 160,000 molecular weight. Aggregation of the dimer was promoted by lower pH values. The enzyme requires pyridoxal 5'-phosphate for activity. The coenzyme appears to be bound in Schiff base linkage as suggested by spectral studies and inhibition by NaBH4. The following sequence was determined for the coenzyme binding site: Val-His-(epsilon-Pxy)Lys-Gln-Gln-Ala-Gly-Gln. The properties of this enzyme are compared with the other biodegradative amino acid decarboxylases that have been isolated from E. coli.     

Pig kidney dopa decarboxylase: inactivation by iodoacetamide and sequence of the carboxyamidomethylcysteine-containing peptide

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.     

Binding to chicken liver lactatedehydrogenase (author's transl)]

Some information about the lactate dehydrogenase NAD binding site has been obtained by working with coenzymes analogs of incomplete molecules. 5'AMP, 5'-ADP, ATP, 5'-c-AMP and 3'(2)-AMP inhibit chicken liver LDH activity competitively with NADH. 5"-AMP and 5'-ADP show a stronger inhibition power than ATP, suggesting that the presence of one or two phosphate groups at the 5' position of adenosine, is essential for the binding of the coenzyme analogs at the enzyme binding site. Ribose and ribose-5'-P do not appear to inhibit the LDH activity, proving that purine base lacking mononucleotides do not bind to the enzyme. 5"-ADPG inhibits LDH activity in the exactly as 5'-ADP, showing that ribose moiety may be replaced by glucose, without considerable effects on the coenzyme analog binding. 2'-desoxidenosin-5'-phosphate proves to be a poorer inhibitor of the LDH activity than 5'-AMP, indicating that an interaction between the--OH groups and the amino-acids of the LDH active center takes place. Nicotinamide does not produce any inhibition effect, while NMN and CMP induce a much weaker inhibition than the adenine analogues, thus indicating a lesser binding capacity to the enzyme. Therefore, the LDH binding site seems to show some definite specificity towards the adenina groups of the coenzyme.     

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.     

The reaction of amino-oxyacetate with pyridoxal phosphate-dependent enzymes.

The carbonyl reagent amino-oxyacetate is frequently used in metabolic studies to inhibit individual pyridoxal phosphate enzymes. The reaction of this compound with three such enzymes, aspartate transaminase, 4-aminobutyrate transaminase and dopa (3,4-dihydroxyphenylalanine) decarboxylase, was studied to determine the extent to which the inhibition is reversible and the rates at which it takes place. Reactions were followed by observing changes in the absorption spectra of the bound coenzyme and by measuring loss of enzyme activity. The reactions with aspartate transaminase and aminobutyrate transaminase were not rapidly reversible and had second-order rate constants (21 degrees C) of 400 M-1.s.1 and 1300 M-1.s-1 respectively and all all concentrations studied showed the kinetics of a simple bimolecular reaction. The reaction with 4-aminobutyrate transaminase could not be reversed and that with aspartate transaminase could only be reversed significantly by addition of cysteinesulphinate to convert the enzyme into its pyridoxamine form. The first-order rate constant (21 degrees C) for the reverse reaction was 4 X 10(-5)s-1. Dopa decarboxylase inhibition by amino-oxyacetate was more rapid and more readily reversible, but measurements of rate and equilibrium constants were not obtained for this enzyme.     

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.     

Microdialysis Monitoring of 3,4-Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 1015 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a 10⁻⁴ mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α-methyl- p -tyrosine. Systemic administration of the α₂-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.     

Effect of administration of 5-hydroxytryptophan and an inhibitor ofl-aromatic amino acid decarboxylase on glucose metabolism in rat brain

The effect of 5-hydroxytryptophan (5-HTP)—the precursor of serotonin (5-hydroxytryptamine, 5-HT)—and of an inhibitor,N-(dl-seryl)-N-(2,3,4-trihydroxybenzyl)hydrazine (Ro4-4602), ofl-aromatic amino acid decarboxylase on the metabolism of glucose to amino acids in brain tissue was investigated. Labeled glucose (20 Ci, 0.24 mg in 0.2 ml 0.9% saline) was injected intravenously into fed rats pretreated with Ro4-4602 (50 mg/kg intraperitoneally) either alone or in combination with 5-HTP (30 mg/kg intravenously) or with the appropriate vehicle. After the injection of Ro4-4602 plus 5-HTP, the concentrations of 5-HT and 5-HTP in brain were increased, but the increase of 5-HTP that Ro4-4602 slightly inhibits the reaction of decarboxylation in the brain, although at the dose used the drug is usually considered to act only peripherally. After administration of Ro4-4602 alone or combined with 5-HTP, the concentration of glucose in plasma was not significantly increased. However, the concentration of glucose in brain was markedly increased with such treatments. The administration of Ro4-4602 alone or combined with 5-HTP reduced the flux of¹⁴C from labeled glucose to amino acids in brain. The concentrations of amino acids in brain were little changed by these treatments.     

Serotonin Agonists Reduce Dopamine Synthesis in the Striatum Only when the Impulse Flow of Nigro-Striatal Neurons Is Intact

The effects of 5-methoxy-N, N-dimethyltryptamine (5-MeO-DMT) and m-chlorophenylpiperazine (CPP), two 5-hydroxytryptamine (5-HT, serotonin) agonists, on the accumulation of 3,4-dihydroxyphenylalanine (DOPA] were studied in the striatum of rats treated with gamma-butyrolactone (GBL). Unlike 2 mg/kg i.p. apomorphine, neither 5 mg/kg i.p. 5-MeO-DMT nor 2.5 mg/kg i.p. CPP significantly reduced the GBL-induced increase in DOPA accumulation in the striatum. 5-MeO-DMT and CPP significantly reduced DOPA accumulation in animals that had received the aromatic amino acid decarboxylase inhibitor Ro 4-4602 but not GBL. 5-HT (10 micrograms in 0.5 microliter) injected in the substantia nigra, pars compacta, like GBL, significantly increased Ro 4-4602-induced accumulation of DOPA in the striatum. The data indicate that 5-HT agonists can reduce 3,4-dihydroxyphenylethylamine (DA, dopamine) synthesis in the striatum of rats only when the impulse flow of DA neurons is intact. An indirect effect through mechanisms controlling DA synthesis in the striatum, for instance cholinergic and GABA-ergic neurons, is suggested.     

Characteristics of Dihydroxyphenylalanine/5-Hydroxytryptophan Decarboxylase Activity in Brain and Liver of Cat

Abstract: Dihydroxyphenylalanine/5-hydroxytryptophan (DOPA/5-HTP) decarboxylase activity varied widely in different parts of the CNS, being highest in the neostriatum and lowest in the frontal cortex. The addition of 2.5 μ m -pyridoxal 5'-phosphate (PLP), the coenzyme, increased enzyme activity in brainstem and liver, while higher concentrations led to a decrease in activity. In brainstem, the addition of 1000 μ m PLP shows activity similar to that obtained without exogenous PLP. The effects of different monoamine oxidase (MAO) inhibitors on decarboxylase activity were demonstrated. Iproniazid phosphate and harmaline significantly decreased the decarboxylation in liver and brainstem, while pargyline inhibited only liver decarboxylation. Some decarboxylase inhibitors such as RO4–4602 and α-methyl DOPA, as well as piribedil, a dopaminergic receptors agonist, were added in vitro to measure their action on decarboxylase with or without exogenous PLP or with double concentrations of substrate (5-HTP). Piribedil (5000 μ m ) affected the enzymic reaction and triggered a higher inhibition in liver. Inhibition in brainstem needed less RO4–4602 (50 μ m ) than in liver (300 μ m ). Addition of PLP did not reverse this inhibition, while doubling the concentration of 5-HTP nullified the inhibitory effect in liver only. Inhibition induced by α-methyl DOPA (5 μ m ) was easily reversed by doubling the concentration of substrate. However, the presence of exogenous PLP restored the enzymic activity in liver only. We conclude from this work thus that the enzyme can decarboxylate its substrate without exogenous PLP, that MAO inhibitors might inhibit decarboxylase activity, and that decarboxylase inhibitors react differently when brain and liver are used as enzymic source. PLP seems to act as a protective agent on the active site of the enzyme in the brainstem and preferentially with the substrate in the liver.     

Novel inhibition of porcine pepsin by a substituted piperidine. Preference for one of the enzyme conformers

Pepsin inhibition by 3-alkoxy-4-arylpiperidine (substituted piperidine; (3R,4R)-3-(4-bromobenzyloxy)-4-[4-(2-naphthalen-1-yl-2-oxo-ethoxy)phenyl]piperidine) has been studied using steady-state kinetic and pre-equilibrium binding methods. Data were compared with pepstatin A, a well known competitive inhibitor of pepsin. Steady-state analysis reveals that the substituted piperidine likewise behaves as a competitive inhibitor. Pre-equilibrium binding studies indicate that the substituted piperidine can displace a fluorescently labeled statine inhibitor from the enzyme active site. Simulation of the stopped-flow fluorescence transients provided estimates of the K(d) values of 1.4 +/- 0.2 microm and 39 +/- 2 nm for the piperidine and the fluorescently labeled statine, respectively. The effects of combinations of these two inhibitors resulted in a series of parallel lines when plotted by the method of Yonetani and Theorell (Yonetani, T., and Theorell, H. (1964) Arch. Biochem. Biophys. 106, 234-251), suggesting that the two inhibitors bind in a mutually exclusive fashion to pepsin. Fitting of the entire data set to the appropriate equation yielded an alpha factor of 8 +/- 1. The magnitude of this factor ( infinity > alpha > 1) can be explained by a conformational distinction between the enzyme species that bind each inhibitor. The effects of pH on the inhibition constants for pepstatin A and the substituted piperidine also suggest that the inhibitors bind to distinct conformational forms of the enzyme. No inhibition by the piperidine was observed at acidic pH, while pepstatin A inhibition is maximal at low pH values. Inhibition by the piperidine was maximal when a group with pK 4.8 +/- 0.2 was deprotonated and another group with pK 5.9 +/- 0.2 was protonated. Most likely these two groups are the catalytic aspartates with perturbed ionization properties as a result of a significant and unique conformational change. Taken together, these data suggest that the enzyme can readily interconvert between two conformers, one capable of binding substrate and pepstatin A and the other capable of binding the substituted piperidine.     

Structure-reactivity probes for active site shapes of cholesterol esterase by carbamate inhibitors.

4,4'-Biphenyl-di-N-butylcarbamate (1), (S)-1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (S-2), (S)-1, 1'-bi-2-naphthyl-2-N-butylcarbamate-2'-butyrate (S-3), 2, 2'-biphenyl-di-N-butylcarbamate (4), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-octylcarbamate (5), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-phenylcarbamate (6), 2, 2'-biphenyl-2-N-butylcarbamate-2'-butyrate (7), 2, 2'-biphenyl-2-N-butylcarbamate-2'-ol (8), 2, 2'-biphenyl-2-N-octylcarbamate-2'-ol (9), (R)-1, 1'-bi-2-N-naphthyl-2-butylcarbamate-2'-ol (R-10), and glyceryl-1,2, 3-tri-N-butylcarbamate (11) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1-3 and 7-10 are the first alkyl chain and esteratic binding site-directed irreversible inhibitors due to the fact that the reactivity of the enzyme is protected by the irreversible inhibitor, trifluoroacetophenone in the presence of these carbamates. Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one of the carbamyl groups of the inhibitor molecule covalently interacts with the first alkyl chain binding site of the enzyme while the other carbamyl group of the inhibitor molecule exposes outside the active site. With near orthogonal conformations at the pivot bond of biaryl groups, one carbamyl group of carbamates S-2, S-3, and R-10 covalently binds to the first alkyl chain binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site probably due to the orthogonal conformations. Carbamates 4-9 and 11 are very potent inhibitors for the enzyme probably due to the fact that all these molecules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains binding sites of the enzyme. Although, carbamates 4-6 and 11 are irreversible inhibitors of cholesterol esterase, the enzyme is not protected but further inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4-6 and 11 covalently bind to the first alkyl chain binding site of the enzyme by one of the carbamyl groups and may also bind to the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable pi-pi interaction between Phe 324 at the second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8-10 are more potent than carbamates S-2, 4, and 5 probably due to the fact that the inhibitor molecules interact with the second alkyl chain binding site of the enzyme through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site.     

Effect of neurotropic agents on total pyridoxal phosphate and on the activity of the decarboxylose of aromatic amino acids as well as of other pyridoxal phosphate-dependent enzymes in rat brains

—(1) Rats received single intraperitoneal injections of various neuroactive chemicals in order to compare the changes of gross behaviour and the level of pyridoxal phosphate as well as the activity of the decarboxylase of aromatic amino acids, of glutamate decarboxylase and of tyrosine transaminase in the brain. (2) The majority of excitatory agents tested (i.e. convulsives like amino-oxyacetate, thiosemicarbazide, pentylenetetrazol and oxotremorine; stimulants such as amphetamine, theophylline and methylphenidate; the amphetamine-like monoamine oxidase inhibitor tranylcypromine as well as the classical monoamine oxidase inhibitor iproniazid when combined with the monoamine releaser Ro 4-1284) caused a decrease in aromatic decarboxylase activity which was coexistent with maximal changes of gross behaviour and partly preceded the latter. The level of pyridoxal phosphate was only partially parallel. As an exception, depression of aromatic decarboxylase was lacking after cocaine (which reduced pyridoxal phosphate only), atropine, the hallucinogens lysergic acid diethylamide and mescaline as well as the combination of the dopamine precursor l -DOPA and the aromatic decarboxylase inhibitor Ro 4-4602. (3) Depression of obvious central nervous functions was almost regularly accompanied and in part preceded by increase of DCA activity (i.e. with the anaesthetics pentobarbitone, diethyl ether and chloroform, the neuroleptics chlorpromazine, haloperidol, reserpine and the benzoquinolizine Ro 4-1284 as well as the tranquillizers diazepam and chlordiazepoxide). Pyridoxal phosphate was increased during or after maximal behavioural changes by pentobarbitone and chlorpromazine only. As an exception, activation of aromatic decarboxylase was absent after morphine. (4) The activity of glutamate decarboxylase was significantly reduced by thiosemicarbazide only, whereas a distinct increase in enzyme activity was exclusively observed after atropine. (5) Tyrosine transaminase activity was significantly diminished by amino-oxyacetate only and showed a late increase after tranylcypromine. (6) It is concluded that there is an inverse relationship, in the majority of neuroactive chemicals tested, between changes of gross behaviour and cerebral aromatic decarboxylase activity. Thereby, the latter is neither regularly related to corresponding variations of the total cerebral pyridoxal phosphate nor to hitherto described alterations of the monoamine turnover nor to effects on other vitamin B₆-dependent enzymes.     

Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds

The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Limited tryptic proteolysis of pig kidney 3,4-dihydroxyphenylalanine decarboxylase

Pig 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.     

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.     

Histamine formation in rat brain in vivo: effects of histidine loads

Abstract— Administration of l -histidine at the rate of 500 mg/kg induced an increase of nearly 50 per cent in the level of histamine in rat brain which lasted several hours. The augmentation of histamine level was not significant 3 h after lower doses or after d -histidine α-methyl DOPA and Ro 4-4602 neither affected the cerebral level of histamine nor its elevation induced by l -histidine. Brocresine, a known histidine decarboxylase inhibitor not only prevented the effect of histidine load but also induced a prompt fall in the amine level. These results confirm those from earlier experiments in vitro indicating that histamine synthesis in rat brain depends on a specific decarboxylase (EC 4 , 1.1.22) which is not normally saturated by the endogenous level of its substrate. When histamine levels were enhanced by histidine treatment, histidine decarboxylase activity, as evaluated on hypothalamus homogenates, was significantly reduced; intracisternal administration of cycloheximide, an inhibitor of protein synthesis, had similar effects. On the other hand, enzyme activity was not altered by the addition of histamine to hypothalamus homogenates. These results are compatible with the existence of a regulation mechanism of histidine decarboxylase involving repression by its end-product.