Phenylalanine ammonia-lyase: A model for the cooperativity kinetics induced by d- and l-phenylalanine

A preparation of l-phenylalanine ammonia-lyase (EC 4.1.3.5.) from soybean (Glycine max L. cv. Kanrich) showed negative cooperativity with respect to l-phenylalanine and competitive inhibition by d-phenylalanine. A two-protomer partially concerted model for inhibition kinetics is described. If cooperativity is associated with ligand binding but not k_cat, plots of v against log [S] at constant [I] are symmetrical. Such curves may be fitted by graphical or iterative least-squares methods. The experimental results conform to this restricted model. The three-substrate and three-inhibitor dissociation constants were estimated by a stepwise procedure. For substrate only the first and second dissociation constants were 12 and 78 μm, respectively, with a symmetry point value of 30.5 μm. To a first approximation, site occupancy determines the cooperativity. As d- and l-phenylalanine produce equivalent effects, they are assumed to pack into the same induced space. As ligand binding at one site has little influence on the relative d:l binding at the other and does not influence k_cat, cooperativity probably reflects changes in regions remote from the active site such as the interface between the protomers. The regulatory range in [S] of the enzyme in vivo may be indicated by the linearity range of the semilog plot for the isolated enzyme. The observed range corresponds to a 100-fold change in [S] compared to a 10-fold change for Michaelis-Menten kinetics.     

A high-throughput assay for screening l- or d-amino acid specific aminotransferase mutant libraries

Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in k_cat/K_M of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.     

Solvolysis of charged active esters of carboxylic acids with cyclo (l- or d-Glu-l-His)

Cyclic dipeptide cyclo(l- or d-Glu-l-His) carrying an anionic site and a nucleophilic site has been synthesized and used as a catalyst for the solvolysis of cationic esters in aqueous alcohols. In the solvolysis of 3-acyloxy-N-trimethylanilinium iodide (S⁺_n, n = 2 and 10) and Cl^?H₃N⁺(CH₂)₁₁COOPh(NO₂), no efficient nucleophilic catalysis was observed. On the other hand, in the solvolysis of Gly-OPh(NO₂)·HCl, Val-OPh(NO₂)·HCl and Leu-OPh(NO₂)·HCl a very efficient general base-type catalysis by cyclo(l-Glu-l-His) was observed. In particular, with the latter two substrates the catalysis by cyclo(l-Glul-His) was more efficient than that by imidazole, although the catalysis was not enantiomer-selective. The diastereomeric cyclic dipeptide cyclo(d-Glu-l-His) was almost inactive under the same conditions. Confomation of cyclo(l- or d-Glu-l-His) in aqueous solution was investigated and the structure/catalysis relationship is discussed.     

Morphology and structure of γ-benzyl-l-glutamate copolymerized with l-phenylalanine, l-valine and l-alanine

Observation of random copolypeptides of γ-benzyl-l-glutamate with l-phenylalanine, l-valine and l-alanine was carried out in an electron microscope with samples cast from dilute solution. The relationship between the morphology and the molecular conformation in solution was studied with mixed solvents composed of chloroform and trifluoroacetic acid; these show a preference for α-helix and random coil, respectively. From the solutions in which molecules take α-helical conformation, fibrous films of nematic structure were formed. From random coil solutions discrete precipitates with folded molecules such as lamellar single crystals, piles of lamellae and structureless particles were formed. A copolypeptide containing l-valine in sufficiently large quantity to form β-structure also showed a variation in morphology with solvent, from films to discrete precipitates. It is suggested that the change in stiffness of the molecules contributes to the morphological variation.     

The orientation of d-β-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane

d-β-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and ‘inside-out’ submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A₂ digestion than from mitochondria. Conclusive evidence that d-β-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome $\text{c}$. The arrangement of d-β-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that d-β-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.     

A new fluorescence assay for dipeptidyleptidase IV using tripeptide l-prolyl-l-prolyl-l-alanine as substrate

We have developed a new fluorescence assay for dipeptidylpeptidase IV using a tripeptide, l-prolyl-l-prolyl-l-alanine, which might be one of the potential natural substrates. The principle of the assay is based on the measurement of fluorescent adduct between alanine liberated from the tripeptide by enzymatic hydrolosis and o-phthaldialdehyde in the presence of 2-mercaptoethanol in aqueous alkaline medium. This new assay is sensitive enough to measure the enzyme activity in as little as 0.01 μl of human serum and in crevicular fluid obtained from human gingival sulcus. The K_m value for the tripeptide was 1.7 · 10^?5 M which is less than one-tenth of that obtained with other chromogenic or fluorogenic substrates. The interference by serum was overcome by simply incorporating the same amount of serum in the standards.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

K+- and Na+-gradient-dependent transport of l-phenylalanine by mouse intestinal brush border membrane vesicles

In the presence of an Na⁺- or a K⁺-gradient ($\text{outside > inside}$), l-phenylalanine uptake exhibited an overshoot phenomenon indicating active transport. The amplitudes of the overshoots were increased by increasing either Na⁺ or K⁺ concentrations in the incubation media, indicating that binding alone cannot account for the K⁺ effect. The K⁺-induced overshoot is not due to the presence of a membrane potential alone, as a gradient of choline chloride failed to produce it. Li⁺ could also substitute for Na⁺ though less potent than Na⁺ in inducing an overshoot. Uptake of l-leucine also showed Na⁺- and K⁺-effects and l-leucine and l-alanine could inhibit the Na⁺- and K⁺-overshoots obtained with phenylalanine. These results lead us to postulate the presence of a carrier for neutral amino acids dependent on monovalent cation with higher affinity for Na⁺ in mouse intestine. The Na⁺- and K⁺-driven active transport of l-phenylalanine were shown to be dependent on the presence of a membrane potential, as short-circuiting the membrane with FCCP reduced the amplitude of the overshoots seen with both ions. However, substitution of Cl^? by more lipophilic anions (NO₃^?, SCN^?) produced an inhibition of uptake. A preliminary analysis of the interrelations between Na⁺ and K⁺ for l-phenylalanine uptake showed complex interactions which can be best explained by mutual competition for a common carrier at both sides of the membrane. These results suggest the presence of a new transport system or a variant of an ASC-type system for l-phenylalanine (and neutral amino acids) in the mouse intestine. However, our studies do not rule out the possible involvement of more than one system for neutral amino acid uptake.     

Synthesis and conformational analysis of cyclo-TRI[l-valyl-d-hexahydromandelyl]

The synthesis of the cyclo-hexadepsipeptide [l-valyl-d-hexahydromandelyl]₃ is described. Examination of this macrocyclic compound by 220-MHz nuclear magnetic resonance spectroscopy shows that symmetrical conformations are stabilized in strongly polar solvents (trifluoroacetic acid, acetonitrile), whereas asymmetric conformations are preferred in nonpolar or slightly polar media such as carbon tetrachloride, chloroform, cyclohexane, and benzene.From analysis of the temperature dependence of the chemical shift and of the coupling constants, together with conformational energy calculations, a model is proposed for the preferred conformation of this molecule in nonpolar solvents.     

Studies on the effect of l-3,4-dehydroproline on collagen synthesis by chick embryo polysemes

Various proline analogs have been tested in vitro for their ability to inhibit the enzymatic aminoacylation of tRNA by proline. Of these, l-3,4-dehydroproline is the most potent inhibitor. This inhibition is competitive; the K_i is 100 μm. It was shown that l-3,4-dehydroproline can serve as substrate in the aminoacylation reaction. However, the incorporation of radioactivity from l-3,4-[¹⁴C]dehydroprolyl-tRNA into protein occurs at one-fifth the rate observed for l-prolyl-tRNA. The addition of l-3,4-dehydroproline in vitro inhibits the synthesis of collagen to a greater extent than non-collagen protein.     

Sodium binding to d-glucuronate residues: Crystal structure of sodium d-glucuronate monohydrate

Three-dimensional X-ray diffraction data were used to determine the crystal structure of sodium β-d-glucuronate monohydrate, a model system for investigating the factors involved in the binding of sodium ions to d-glucuronate residues of glycosaminoglycans. Crystals of the salt are monoclinic, space group P2₁, with a = 9.206(3) Å, b = 7.007(2) Å, c = 7.378(3) Å, β = 96.84(3)°, and Z = 2. Intensity data for 858 reflections were measured with an automated diffractometer. A trial structure, obtained by direct methods, was refined by least squares to R = 0.035. An outstanding feature of the crystal packing is the interaction of d-glucuronate anions with sodium ions. The sodium ion is coordinated to three symmetry-related $\text{d}$-glucuronate anions and to one water molecule. The d-glucuronate anion binds sodium cations through the three following sites: one that involves a carboxyl oxygen atom combined with ring oxygen O-5; one that includes a single carboxyl oxygen atom, and one composed of the O-3–O-4 pair of hydroxyl groups.     

ATP binding to brain l-glutamate decarboxylase: A study by affinity chromatography

The binding of ATP to brain l-glutamate decarboxylase (GAD) was studied by means of ATP-agarose chromatography, utilizing partially purified GAD from mouse brain after DEAE-cellulose chromatography and ammonium sulfate fractional precipitation. GAD was found to bind with a high affinity to the ATP-agarose with the ATP molecule linked to the beaded agarose through the N⁶-amino group. Agarose with ATP attached through the ribosyl hydroxyls was totally ineffective to bind the enzyme. GAD bound to the immobilized ATP could be dissociated by free ATP (10–50 mM), but not by ADP at a concentration as high as 100 mM. Mg²⁺ was not a required factor for the binding. The enzyme binding to the ATP-agarose occurred under a saturating concentration (50 μM) of pyridoxal 5′-phosphate (PLP). Moreover, GAD bound to the ATP-agarose was not dissociated by PLP even at 1.0 mM, indicating no competition of PLP with ATP for the same binding site on the enzyme. Kinetic characterization showed that binding of ATP raised the K_m of the enzyme for PLP. Our approach provides direct evidence that there is a specific binding site on GAD for ATP, which is distinct from the binding site for PLP.     

Studies on aroyl- and aryl-hydrazide derivatives from d-glycero-d-gulo-heptono-1,4-lactone

A series of aroyl- and aryl-hydrazide derivatives was prepared from d-glycero-d-gulo-heptono-1,4-lactone (1). The reactivity of the NH proton in these hydrazides, in terms of their dissociation constants (pK_a), was determined from their electronic spectra, and correlated to the Hammett σ values of the substituents. Comparable reactivities of the NH protons for the compounds, and the effect of the substituent, were studied by n.m.r. spectroscopy. Decomposition of the aroylhydrazides with copper(II) sulfate or nitrous acid resulted in the regeneration of 1.     

Transport of l-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of l-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris·HCl of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in part buffered saline. l-Methionine is actively transported in WI38 by saturable, chemically specific mechanisms which are temperature, pH and, in part, Na⁺ dependent, and are reactive with both l- and d-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent K_M of 21.7 μM and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent K_M of 547 μM and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na⁺- free media or after treatment with ouabain suggested that system 1 is Na⁺ independent and that system 2 is Na⁺ dependent. Preloading of cells with unlabeled l-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled l- or d-methionine or l-phenylalanine, but not in the presence of l-arginine. l-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

Bioanalytical method for the simultaneous determination of d- and l-serine in human plasma by LC/MS/MS

d-Serine is an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors. Plasma concentrations of d-serine and the ratio of d-serine to total serine may be used as clinically-translatable biomarkers in NMDA receptor-related disease. We developed a highly sensitive and specific method using high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the d- and l-isomers of serine in human plasma. Since d- and l-serine are endogenous components, phosphate buffered saline was used as the surrogate matrix. d- and l-serine in human plasma and PBS were treated by cationic exchange solid phase extraction. d-Serine (m/z 106.1 > 60.0), l-serine (m/z 106.1 > 60.1) and dl-serine-d₃ (m/z 109.1 > 63.0) were detected using a multiple reaction monitoring. The enantiomer separation of d- and l-serine was successfully achieved without any derivatization step using tandemly-arranged and ice-cold CROWNPAK CR-I(+) columns with an isocratic mobile phase comprised of 0.3% trifluoroacetic acid in 10% acetonitrile. The standard curves were linear throughout the calibration range with 0.01–10 μg/mL (d-serine) and 0.1–100 μg/mL (l-serine), respectively. Intra-day and inter-day precision and accuracy of the quality control samples were within relative standard deviations of less than 15%. The endogenous concentrations of d- and l-serine in human plasma were 0.124–0.199 and 7.97–13.1 μg/mL, respectively.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.