Transport of diamines by Enterococcus faecalis is mediated by an agmatine-putrescine antiporter.

Enterococcus faecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway, uptake of arginine and excretion of ornithine are mediated by an arginine-ornithine antiport system. The translocation of agmatine was studied in whole cells grown in the presence of arginine, agmatine, or glucose. Rapid uncoupler-insensitive uptake of agmatine was observed only in agmatine-grown cells. A high intracellular putrescine pool was maintained by these cells, and this pool was rapidly released by external putrescine or agmatine but not by arginine or ornithine. Kinetic analysis revealed competitive inhibition for uptake between putrescine and agmatine. Agmatine uptake by membrane vesicles was observed only when the membrane vesicles were preloaded with putrescine. Uptake of agmatine was driven by the outwardly directed putrescine concentration gradient, which is continuously sustained by the metabolic process. Uptake of agmatine and extrusion of putrescine by agmatine-grown cells of E. faecalis appeared to be catalyzed by an agmatine-putrescine antiporter. This transport system functionally resembled the previously described arginine-ornithine antiport, which was exclusively induced when the cells were grown in the presence of arginine.     

Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi.

Trypanosoma cruzi was found to release 14CO2 from radiolabeled arginine, and this effect was inhibited by either DL-alpha-difluoromethylarginine or monofluoromethylagmatine, both specific inhibitors of arginine decarboxylase (ADC). Furthermore, agmatine, which can be derived metabolically only by ADC-mediated arginine decarboxylation, was produced when T. cruzi was incubated with radiolabeled arginine, and agmatine production was inhibited in the presence of DL-alpha-difluoromethylarginine. These results constitute direct biochemical evidence for the presence in T. cruzi of ADC, an enzyme that does not occur in mammalian cells.     

Progress-curve equations for reversible enzyme-catalysed reactions inhibited by tight-binding inhibitors.

The rate equation for a tight-binding inhibitor of an enzyme-catalysed first-order reversible reaction was used to derive two integrated equations. One of them covers the situations in which competitive, uncompetitive or non-competitive inhibition occurs and the other refers to the special non-competitive case where the two inhibition constants are equal. For these equations, graphical and non-linear regression methods are proposed for distinguishing between types of inhibition and for calculating inhibition constants from progress-curve data. The application of the non-linear regression to the analysis of stimulated progress curves in the presence of a tight-binding inhibitor is also presented. The results obtained are valid for any type of 'dead-end'-complex-forming inhibitor and can be used to characterize an unknown inhibitor on the basis of progress curves.     

Inhibition of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle

Using stopped flow methods, we have measured the steady state rate constants and the inhibition by N3- and I- of the hydration of CO2 catalyzed by carbonic anhydrase III from cat muscle. Also, using fluorescence quenching of the enzyme at 330 nm, we have measured the binding of the sulfonamide chlorzolamide to cat carbonic anhydrase III. Inhibition by the anions was uncompetitive at pH 6.0 and was mixed at higher values of pH. The inhibition constant of azide was independent of pH between 6.0 and 7.5 with a value of KIintercept = 2 X 10(-5) M; the binding constant of chlorzolamide to cat carbonic anhydrase III was also independent of pH in the range of 6.0 to 7.5 with a value Kdiss = 2 X 10(-6) M. Both of these values increased as pH increased above 8. There was a competition between chlorzolamide and the anions N-3 and OCN- for binding sites on cat carbonic anhydrase III. The pH profiles for the kinetic constants and the uncompetitive inhibition at pH 6.0 can be explained by an activity-controlling group in cat carbonic anhydrase III with a pKa less than 6. Moreover, the data suggest that like isozyme II, cat isozyme III is limited in rate by a step occurring outside the actual interconversion of CO2 and HCO3- and involving a change in bonding to hydrogen exchangeable with solvent water.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.     

Carbonyl sulfide inhibition of CO dehydrogenase from Rhodospirillum rubrum

Carbonyl sulfide (COS) has been investigated as a rapid-equilibrium inhibitor of CO oxidation by the CO dehydrogenase purified from Rhodospirillum rubrum. The kinetic evidence suggests that the inhibition by COS is largely competitive versus CO (Ki = 2.3 microM) and uncompetitive versus methylviologen as electron acceptor (Ki = 15.8 microM). The data are compatible with a ping-pong mechanism for CO oxidation and COS inhibition. Unlike the substrate CO, COS does not reduce the iron-sulfur centers of dye-oxidized CO dehydrogenase and thus is not an alternative substrate for the enzyme. However, like CO, COS is capable of protecting CO dehydrogenase from slow-binding inhibition by cyanide. A true binding constant (KD) of 2.2 microM for COS has been derived on the basis of the saturable nature of COS protection against cyanide inhibition. The ability of CO, CO2, COS, and related CO/CO2 analogues to reverse cyanide inhibition of CO dehydrogenase is also demonstrated. The kinetic results are interpreted in terms of two binding sites for CO on CO dehydrogenase from R. rubrum.     

Hypotensive effect of agmatine, arginine metabolite, is affected by NO synthase

The metabolites of arginine were recently shown to be involved in cardiovascular control. The study addresses the general cardiovascular response of anaesthetized rats to agmatine, a decarboxylated arginine. The relation between two arginine metabolic pathways governed by arginine decarboxylase and nitric oxide synthase was investigated. Intravenous administration of agmatine 30 and 60 microM/0.1 ml saline elicited remarkable hypotension of 42.6+/-4.6 and 70.9+/-6.5 mm Hg, respectively. The hypotension was characterized by long duration with half-time of return 171.6+/-2.9 and 229.2+/-3.8 s, respectively. The time of total blood pressure BP recovery was about 10 min. Dose-dependent relaxation to agmatine was also found in aorta rings in vitro. Both doses of agmatine administered 60-180 min after NO synthase inhibition L-NAME 40 mg/kg i.v. caused greater hypotension 59.0+/-7.6 and 95.8 8.8 mm Hg P<0.01 both compared to animals with intact NO synthase, but this was accompanied by a significant shortening of the half-time of BP return. If agmatine was administered to hypertensive NO-deficient rats treated with 40 mg/kg/day L-NAME for 4 weeks, similar significant enhancement of hypotension was observed at both agmatine doses, again with a significant shortening of half-time of BP return. It can be summarized that the long-lasting hypotension elicited by agmatine was amplified after acute or chronic NO synthase inhibition, indicating a feedback relation between the two metabolic pathways of arginine.     

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. II. Substrate interaction and product inhibition studies of flavonol 3-, 6-, and 4'-O-methyltransferases

The steady-state kinetic behavior of three position-specific O-methyltransferases (3-, 4'-, and 6-OMTs) was compared with reference to substrate inhibition patterns in Chrysosplenium americanum. The 6-OMT was severely inhibited by the flavonoid substrate at concentrations close to Km, whereas the other two enzymes were less affected by their respective flavonoid substrates. Substrate interaction kinetics for the 6-OMT gave converging lines consistent with a sequential binding mechanism, whereas the data generated for the 3- and 4'-OMTs could be fitted to the equation for a ping-pong mechanism or to that of a sequential binding mechanism where Kia was much smaller Ka. More information on the mechanism of reaction was obtained from product inhibition studies. The three enzymes exhibited competitive inhibition patterns between S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH), whereas other patterns were either noncompetitive or uncompetitive. The steady-state kinetic properties of the 3-, 4'-, and 6-OMTs were consistent with a sequential ordered reaction mechanism, in which SAM and SAH were leading reaction partners and included an abortive EQB complex. Product inhibition constants were sufficiently low to suggest that these may be important in regulating the pathway of polymethylated flavonoid synthesis. It was suggested that due to their greater sensitivity to inhibition by SAH, the OMTs involved in earlier steps of the methylation sequence may regulate the rate of synthesis of final products in Chrysosplenium.     

Screening of substrate analogs as potential enzyme inhibitors for the arginine kinase of Trypanosoma cruzi

Arginine kinase catalyzes the transphosphorylation between phosphoarginine and ADP. Phosphoarginine is involved in temporal ATP buffering and inorganic phosphate regulation. Trypanosoma cruzi arginine kinase phosphorylates only L-arginine (specific activity 398.9 x mUE-min(-1) x mg(-1)), and is inhibited by the arginine analogs, agmatine, canavanine, nitroarginine, and homoarginine. Canavanine and homoarginine also produce a significant inhibition of the epimastigote culture growth (79.7% and 55.8%, respectively). Inhibition constants were calculated for canavanine and homoarginine (7.55 and 6.02 mM, respectively). In addition, two novel guanidino kinase activities were detected in the epimastigote soluble extract. The development of the arginine kinase inhibitors of T. cruzi could be an important feature because the phosphagens biosynthetic pathway in trypanosomatids is different from the one in their mammalian hosts.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Kinetic modelling and simulation of laccase catalyzed degradation of reactive textile dyes

A kinetic model based on Michaelis-Menten equation was developed to simulate the dye decolourisation of Reactive Black 5 (RB5), Reactive Blue 114 (RB114), Reactive Yellow 15 (RY15), Reactive Red 239 (RR239) and Reactive Red 180 (RR180) dyes by commercial laccase. The unusual kinetic behavior of some of these reactions suggests that the kinetic model must consider the activation of the laccase-mediator system. Several reactions at different concentrations of each dye were performed in batch reactors and time courses were obtained. A LSODE code to solve the differential equation obtained from the batch reactor was combined with an optimization Fortran program to obtain the theoretical time courses. The time courses obtained from the developed program were compared with the experimentally obtained ones to estimate the kinetic constants that minimized the difference between them. The close correlation between the predicted and the experimental results seems to support the reliability of the established models.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: the kinetic properties of the enzyme

The kinetic properties for the native forward reaction of pyruvate:NADP+ oxidoreductase from Euglena gracilis were determined. The substrate kinetics gave a pattern of a ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate. The Km values for pyruvate, CoA, and NADP+ were estimated to be 27, 6.6, and 28 microM, respectively, and the Ki value of CoA against pyruvate was 28 microM. CO2 inhibited noncompetitively against pyruvate and NADP+, and uncompetitively against CoA. Acetyl-CoA showed a competitive inhibition with respect to pyruvate and an uncompetitive inhibition with respect to NADP+. NADPH inhibited competitively versus NADP+, noncompetitively versus CoA, and uncompetitively versus pyruvate. The kinetic behavior is consistent with a two-site ping-pong mechanism involving the substrate inhibition. From the kinetic mechanism, it is proposed that the enzyme has two catalytic sites linked by an intramolecular electron-transport chain. One of these is a thiamine pyrophosphate-containing catalytic site which reacts with pyruvate and CoA to form CO2 and acetyl-CoA, and the other site functions in the reduction of NADP+. In contrast, when methyl viologen was used as an artificial one-electron acceptor substituting for NADP+, the reaction gave a pattern characteristic of an octa uni ping-pong mechanism involving a competitive substrate inhibition of CoA against pyruvate.     

Kinetic mechanism of the aliphatic amidase from Pseudomonas aeruginosa.

The kinetic constants for hydrolysis and transfer (with hydroxylamine as the alternate acceptor) of the aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were determined for a variety of acetyl and propionyl derivatives. The results obtained were consistent with a ping-pong or substitution mechanism. Product inhibition, which was pH dependent, implicated an acyl-enzyme compound as a compulsory intermediate and indicated that ammonia combined additionally with the free enzyme in a dead-end manner. The uncompetitive activation of acetamide hydrolysis by hydroxylamine and the observation that the partitioning of products between acetic acid and acetohydroxamate was linearly dependent on the hydroxylamine concentration substantiated these conclusions and indicated that deacylation was at least partially rate limiting. With propionamide as the acyl donor apparently anomalous results, which included inequalities in certain kinetic constants and a hyperbolic dependence of the partition ratio on the hydroxylamine concentration, could be explained by postulating a compulsory isomerisation of the acyl-enzyme intermediate prior to the transfer reaction.     

Lack of arginine decarboxylase in Trypanosoma cruzi epimastigotes

The presence of arginine decarboxylase (ADC) enzymatic activity in Trypanosoma cruzi epimastigotes is still a matter of controversy due to conflicting results published during the last few years. We have investigated whether arginine might indeed be a precursor of putrescine via agmatine in these parasites. We have shown that wild-type T. cruzi epimastigotes cultivated in a medium almost free of polyamines stopped their growth after several repeated passages of cultures in the same medium, and that neither arginine nor omithine were able to support or reinitiate parasite multiplication. In contrast, normal growth was quickly resumed after adding exogenous putrescine or spermidine. The in vivo labelling of parasites with radioactive arginine showed no conversion of this amino acid into agmatine, and attempts to detect ADC activity measured by the release of CO2 under different conditions in T. cruzi extracts gave negligible values for all strains assayed. The described data clearly indicate that wild-type T. cruzi epimastigotes lack ADC enzymatic activity.     

Regulation of urea synthesis by agmatine in the perfused liver: studies with 15N

Administration of arginine or a high-protein diet increases the hepatic content of N-acetylglutamate (NAG) and the synthesis of urea. However, the underlying mechanism is unknown. We have explored the hypothesis that agmatine, a metabolite of arginine, may stimulate NAG synthesis and, thereby, urea synthesis. We tested this hypothesis in a liver perfusion system to determine 1) the metabolism of l-[guanidino-15N2]arginine to either agmatine, nitric oxide (NO), and/or urea; 2) hepatic uptake of perfusate agmatine and its action on hepatic N metabolism; and 3) the role of arginine, agmatine, or NO in regulating NAG synthesis and ureagenesis in livers perfused with 15N-labeled glutamine and unlabeled ammonia or 15NH4Cl and unlabeled glutamine. Our principal findings are 1) [guanidino-15N2]agmatine is formed in the liver from perfusate l-[guanidino-15N2]arginine ( approximately 90% of hepatic agmatine is derived from perfusate arginine); 2) perfusions with agmatine significantly stimulated the synthesis of 15N-labeled NAG and [15N]urea from 15N-labeled ammonia or glutamine; and 3) the increased levels of hepatic agmatine are strongly correlated with increased levels and synthesis of 15N-labeled NAG and [15N]urea. These data suggest a possible therapeutic strategy encompassing the use of agmatine for the treatment of disturbed ureagenesis, whether secondary to inborn errors of metabolism or to liver disease.     

Fatty acid synthetase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from pigeon liver.

The kinetic mechanism of pigeon liver fatty acid synthetase action has been studied using steady state kinetic analysis. Initial velocity studies are consistent with an earlier suggestion that the enzyme catalyzes this reaction by a seven-site ping-pong mechanism. Although the range of substrate concentrations that could be used was limited by several factors, the initial velocity patterns showing the relationship between the substrates acetyl coenzyme CoA, malonyl-CoA, and NADPH appear to be a series of parallel lines, regardless of which substrate is varied at fixed levels of a second substrate. However, two of the substrates, acetyl-CoA and malonly-CoA, apparently exhibit a competitive substrate inhibition with respect to each other, but NADPH shows no inhibition of any kind. Product inhibition patterns suggest that free CoA is competitive versus acetyl-CoA and malonyl-CoA and is uncompetitive versus NADPH, and that NADP+ is competitive versus NADPH and uncompetitive versus acetyl-CoA or malonyl-CoA. These results are consistent with a seven-site ping-pong mechanism with intermediates covalently bound to 4'-phosphopantetheine (part of acyl carrier protein). Double competitive substrate inhibition by acetyl-CoA and malonyl-CoA is consistent with the rate equation derived for the over-all mechanism. The kinetic mechanism developed from these results is capable of explaining the formation of fatty acids from malonyl-CoA and NADPH alone (Katiyar, S. S., Briedis, A. V., and Porter, J. W. (1974) Arch. Biochem. Biophys. 162, 412-420) and also the formation of triacetic acid lactone from either malonyl-CoA alone or acetyl-CoA plus malonyl-CoA.     

Enzymes of agmatine degradation and the control of their synthesis in Streptococcus faecalis.

Streptococcus faecalis ATCC 11700 uses agmatine as its sole energy source for growth. Agmatine deiminase and putrescine carbamoyltransferase are coinduced by growth on agmatine. Glucose and arginine were found to exert catabolite repression on the agmatine deiminase pathway. Four mutants unable to utilize agmatine as an energy source, isolated from the wild-type strain, exhibited three distinct phenotypes. Two of these strains showed essentially no agmatine deiminase, one mutant showed negligible activity of putrescine carbamoyltransferase, and one mutant was defective in both activities. Two carbamate kinases are present in S. faecalis, one belonging to the arginine deiminase pathway, the other being induced by growth on agmatine. These two enzymes have the same molecular weight, 82,000, and seem quite different in size from the kinases isolated from other streptococci.     

Regulation of potential-dependent L-type Ca2+ currents by agmatine. Imidazoline receptors in isolated cardiomyocytes

The main goal of the present work was to study the mechanisms of voltage-gated L-type Ca²⁺ currents regulation by agmatine in isolated cardiomyocytes and to determine whether agmatine is involved in mediating the “arginine paradox”. It was shown that agmatine at concentrations from 200 μM to 15 mM inhibited L-type Ca²⁺ currents in isolated cardiomyocytes in a dose-dependent manner. The selective antagonists of α₂-adrenoceptors (α₂-ARs), yohimbine and rauwolscine, did not modulate the effect of agmatine. In contrast, efaroxan and idazoxan known to antagonize both α₂-ARs and type 1 imidazoline receptors (I₁Rs) decreased the efficiency of agmatine almost twofold. The NO synthase inhibitor 7NI insignificantly influenced the suppressive action of agmatine on L-type Ca²⁺ currents, whereas the protein kinase C inhibitor, calphostin C, markedly reduced the effects of agmatine. Arginine did not affect L-type Ca²⁺ currents in the presence of agmatine and vice versa. These data suggest that agmatine is not involved in mediating the “arginine paradox” and that its effects are not due to the activation of endothelial NO synthase (eNOS) followed by cGMP-dependent inhibition of L-type Ca²⁺ current. Most likely, agmatine acts via I₁Rs coupled with the signaling pathway that involves the activation of protein kinase C. Previously nothing was known about possible localization of I₁Rs in isolated cardiomyocytes. Consistently, we have shown that single cardiomyocytes express the nischarin genes homologous to the IRAS gene, which is considered in the modern literature as the major candidate for the gene encoding I₁Rs. To the best our knowledge, this is the first demonstration of I₁Rs expression at the level of individual cells, including cardiomyocytes.