Aromatic amino acid transport in Yersinia pestis.

The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-Menten-like kinetics with apparent Km values of 6 x 10(-7) to 7.5 x 10(-7) and 2 x 10(-6) M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10(-6) M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 TO8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact alpha amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and tyrosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhydryl reagents or sodium azide. The results on the selective inhibition of aromatic amino acid transport provide additional evidence for multiple transport systems . These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy.     

Formation of aromatic amino acid pools in Escherichia coli K-12

Phenylalanine, tyrosine, and tryptophan were taken up into cells of Escherichia coli K-12 by a general aromatic transport system. Apparent Michaelis constants for the three amino acids were 4.7 x 10(-7), 5.7 x 10(-7), and 4.0 x 10(-7)m, respectively. High concentrations (> 0.1 mm) of histidine, leucine, methionine, alanine, cysteine, and aspartic acid also had an affinity for this system. Mutants lacking the general aromatic transport system were resistant to p-fluorophenylalanine, beta-2-thienylalanine, and 5-methyltryptophan. They mapped at a locus, aroP, between leu and pan on the chromosome, being 30% cotransducible with leu and 43% cotransducible with pan. Phenylalanine, tyrosine, and tryptophan were also transported by three specific transport systems. The apparent Michaelis constants of these systems were 2.0 x 10(-6), 2.2 x 10(-6), and 3.0 x 10(-6)m, respectively. An external energy source, such as glucose, was not required for activity of either general or specific aromatic transport systems. Azide and 2,4-dinitrophenol, however, inhibited all aromatic transport, indicating that energy production is necessary. Between 80 and 90% of the trichloroacetic acid-soluble pool formed from a particular exogenous aromatic amino acid was generated by the general aromatic transport system. This contribution was abolished when uptake was inhibited by competition by the other aromatic amino acids or by mutation in aroP. Incorporation of the former amino acid into protein was not affected by the reduction in its pool size, indicating that the general aromatic transport system is not essential for the supply of external aromatic amino acids to protein synthesis.     

Design of inhibitors for human tissue kallikrein using non-natural aromatic and basic amino acids

We explored the unique substrate specificity of the primary S, subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099-1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positively-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.     

Quinoline-2,4-dicarboxylic acids: synthesis and evaluation as inhibitors of the glutamate vesicular transport system.

Twenty-six quinoline-2,4-dicarboxylic acids (QDC's) were synthesized by a modified Doebner-von Miller pathway and tested as inhibitors against the glutamate vesicular transport (GVT) protein. The QDC's were active as inhibitors with the most potent QDC's found to contain halogens at the 6-/8-position, a hydroxyl at the 8-position, or a tethered aromatic moiety at the 6- or 7-position of the quinoline.     

Inhibition of electron transport and oxidative phosphorylation in plant mitochondria by gladiolic acid and structurally-related aromatic ortho dialdehydes

Gladiolic acid (GA, 4-methoxy-5-methyl-0-phthalaldehyde-3-carboxylic acid), an antifungal aromatic ortho dialdehyde produced by Penicillium gladioli was found to be a potent inhibitor of electron transport and oxidative phosphorylation reactions in sweet potato and mung bean mitochondria. Similar results were also found with the naturally occurring ortho dialdehydes, cyclopaldic acid, quadrilineatin, and flavipin as well as the synthetic dialdehydes, 3-formyl opianic acid and 0-phthalaldehyde. Because of their highly reactive ortho-diformyl grouping, GA and structurally related dialdehydes apparently act as multisite inhibitors affecting electron transport and oxidative phosphorylation (at each coupling site). Gladiolic acid has no uncoupling effect like 2,4-dinitrophenol and does not have the same point of interaction in the energy transfer process as oligomycin. Several "partial" reactions of phosphorylation (Mg+2-DNP-stimulated ATPase; ATP-Pi exchange) were strongly inhibited by the various dialdehydes. Flavipin and quadrilineatin are potent inhibitors (80% at a concentration of 25 microM) of site III phosphorylation. Gladiolic acid and related ortho dialdehydes inactivate the catalytic activity of native cytochrome c in vitro. Lysyl epsilon-NH2 rich cytochrome c may be a major site of GA action in the intact mitochondrion. In view of the high chemical reactivity of the orthodiformyl group, it is suggested that mitochondrial function may be affected by aromatic ortho dialdehydes through a combination of reactions involving cross-linking of amino groups on membrane polypeptides and monofunctional reaction with free amino groups important for enzyme function, including epsilon-NH2 groups on cytochrome c. Cross-linking in mitochondrial membrane systems might affect function by interfering with molecular motion in the operation of the terminal portion of the electron-transport chain. The primary toxicological mode of action of GA and related dialdehydes appears to be due to inhibition of mitochondrial function.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes

Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

Effects of single D-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S.

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.     

S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids.

We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K(i) of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P(1) position.     

Relationship between structure and P-glycoprotein inhibitory activity of dimeric peptides related to the Dmt-Tic pharmacophore

To develop novel inhibitors of P-glycoprotein (P-gp), dimeric peptides related to an opioid peptide containing the Dmt-Tic pharmacophore were synthesized and their P-gp inhibitory activities were analyzed. Of the 30 analogs synthesized, N(α),N(ε)-[(CH(3))(2)Mle-Tic](2)Lys-NH(2) and its D-Lys analog were found to exhibit potent P-gp inhibitory activity, twice that of verapamil, in doxorubicin-resistant K562 cells. Structure-activity studies indicated that the correct hydrophobicity and spacer length between two aromatic rings are important structural elements in this series of analogs for inhibition of P-gp.     

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.     

Biochemical changes in French bean pods infected with Colletotrichum lindemuthianum*

Infection of bean pods with Colletotrichum lindemuthianum leads to the appearance in diffusates of a range of fluorescent and phenolic compounds and of at least two inhibitory compounds. These compounds were found to be absent or in reduced concentration in control diffusates, although the inhibitors were frequently observed to appear at low concentrations without infection. Although sugars and amino acids were found to be released into diffusates, variation in the concentration of these stimulants was found to have little effect on spore germination. Evidence from solvent partition, spectrophotometry and chromatography suggests that the two inhibitors can be tentatively identified with inhibitors previously described from French bean, and both appear to be phenolic substances. It is suggested that inhibitor production may be regarded as part of a general change in aromatic biosynthesis following infection.     

Purification and characterization of a tuberculin-active substance from Mycobacterium bovis BCG

A new tuberculin-active substance, designated TAS-1D3, has been purified from the extract of Mycobacterium bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography involving CM-cellulose, QAE-Sephadex A-25, Sephadex G-100, and Sephadex G-75. TAS-1D3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1D3 is a glycoprotein. The molecular weight of TAS-1D3 was estimated to be 26,000 by gel filtration. In amino acid analysis, TAS-1D3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfur-containing amino acids and aromatic amino acids. Moreover, TAS-1D3 was almost devoid of absorption at around 280 nm. In guinea pigs sensitized with BCG vaccine, the tuberculin activity of TAS-1D3 was about forty times more potent than that of purified protein derivative (PPD).     

Decline in ribonucleic acid and protein synthesis with loss of viability during the early hours of imbibition of rye (Secale cereale L.) embryos.

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids.     

Determination of DOPA,dopamine, DOPAC,epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. By using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxypehnylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). α-Difluoromethyldopa (DFMD) and α-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Maintenance and exchange of the aromatic amino acid pool in Escherichia coli

The pool of phenylalanine, tyrosine, and tryptophan is formed in Escherichia coli K-12 by a general aromatic transport system [Michaelis constant (K(m)) for each amino acid approximately 5 x 10(-7)m] and three further transport systems each specific for a single aromatic amino acid (K(m) for each amino acid approximately 2 x 10(-6)m, reference 3). When the external concentration of a particular aromatic amino acid is saturating for both classes of transport system, the free amino acid pool is supplied with external amino acid by both systems. Blocking the general transport system reduces the pool size by 80 to 90% but does not interfere with the supply of the amino acid to protein synthesis. If, however, the external concentration is too low to saturate specific transport, blocking general transport inhibits the incorporation of external amino acid into protein by about 75%. It is concluded that the amino acids transported by either class of transport system can be used for protein synthesis. Dilution of the external amino acid or deprivation of energy causes efflux of the aromatic pool. These results and rapid exchange observed between pool amino acid and external amino acids indicate that the aromatic pool circulates rapidly between the inside and the outside of the cell. Evidence is presented that this exchange is mediated by the aromatic transport systems. Mutation of aroP (a gene specifying general aromatic transport) inhibits exit and exchange of the small pool generated by specific transport. These findings are discussed and a simple physiological model of aromatic pool formation, and exchange, is proposed.     

Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)