L-lysine Binding Research Articles

Mechanistic studies on the flavin-dependent N6-lysine monooxygenase MbsG reveal an unusual control for catalysis

The mechanism of Mycobacterium smegmatis G (MbsG), a flavin-dependent l-lysine monooxygenase, was investigated under steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, substrate analogs, pH and solvent viscosity effects as mechanistic probes. The results suggest that l-lysine binds before NAD(P)H, which leads to a decrease in the rate constant for flavin reduction. l-lysine binding has no effect on the rate of flavin oxidation, which occurs in a one-step process without the observation of a C4a-hydroperoxyflavin intermediate. Similar effects were determined with several substrate analogs. Flavin oxidation is pH independent while the kcat/Km and kred/KD pH profiles for NAD(P)H exhibit single pKa values of ∼6.0, with increasing activity as the pH decreases. At lower pH, the enzyme becomes more uncoupled, producing more hydrogen peroxide and superoxide. Hydride transfer is partially rate-limiting at neutral pH and becomes more rate-limiting at low pH. An inverse solvent viscosity effect on kcat/Km for NAD(P)H was observed at neutral pH whereas a normal solvent viscosity effect was observed at lower pH. Together, the results indicate a unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation.

A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily: PRODUCTION, FUNCTIONAL CHARACTERIZATION, AND STRUCTURE MODELING

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of L-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of L-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of L-arginine, L-ornithine, L-2,4-diaminobutyric acid, and L-alanine. D-lysine is bound 45 times more weakly than its L-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010-1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for L-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

Ceftaroline Restores Daptomycin Activity against Daptomycin-Nonsusceptible Vancomycin-Resistant Enterococcus faecium

Daptomycin-nonsusceptible vancomycin-resistant Enterococcus faecium (VRE) strains are a formidable emerging threat to patients with comorbidities, leaving few therapeutic options in cases of severe invasive infections. Using a previously characterized isogenic pair of VRE strains from the same patient differing in their daptomycin susceptibilities (Etest MICs of 0.38 mg/liter and 10 mg/liter), we examined the effect of ceftaroline, ceftriaxone, and ampicillin on membrane fluidity and susceptibility of VRE to surface binding and killing by daptomycin and human cathelicidin antimicrobial peptide LL37. Synergy was noted in vitro between daptomycin, ampicillin, and ceftaroline for the daptomycin-susceptible VRE strain, but only ceftaroline showed synergy against the daptomycin-nonsusceptible VRE strain (∼2 log10 CFU reduction at 24 h). Ceftaroline cotreatment increased daptomycin surface binding with an associated increase in membrane fluidity and an increase in the net negative surface charge of the bacteria as evidenced by increased poly-l-lysine binding. Consistent with the observed biophysical changes, ceftaroline resulted in increased binding and killing of daptomycin-nonsusceptible VRE by human cathelicidin LL37. Using a pair of daptomycin-susceptible/nonsusceptible VRE strains, we noted that VRE is ceftaroline resistant, yet ceftaroline confers significant effects on growth rate as well as biophysical changes on the cell surface of VRE that can potentiate the activity of daptomycin and innate cationic host defense peptides, such as cathelicidin. Although limited to just 2 strains, these finding suggest that additional in vivo and in vitro studies need to be done to explore the possibility of using ceftaroline as adjunctive anti-VRE therapy.

Ampicillin Enhances Daptomycin- and Cationic Host Defense Peptide-Mediated Killing of Ampicillin- and Vancomycin-Resistant Enterococcus faecium

We studied an ampicillin- and vancomycin-resistant Enterococcus faecium (VRE) isolate from a patient with endocarditis and bacteremia refractory to treatment with daptomycin (6 mg/kg of body weight) plus linezolid. Blood cultures cleared within 24 h of changing therapy to daptomycin (12 mg/kg) plus ampicillin. We examined the effects of ampicillin on daptomycin-induced growth inhibition and killing, surface charge, and susceptibility to several prototypical host defense cationic antimicrobial peptides. MICs and time-kill curves with daptomycin were assessed in the presence and absence of ampicillin. The impact of ampicillin on surface charge was assessed by flow cytometry and a poly-l-lysine binding assay. The effects of ampicillin preexposures upon VRE killing by five distinct cationic peptides of different structure, charge, origin, and mechanism of action were analyzed using the epidermal cathelicidin LL-37, thrombin-induced platelet microbicidal proteins (tPMPs), and a synthetic congener modeled after tPMP microbicidal domains (RP-1), human neutrophil peptide-1 (hNP-1), and polymyxin B (bacteria derived). Fluoroscein-Bodipy-labeled daptomycin was used to evaluate daptomycin binding to VRE membranes in the presence or absence of ampicillin. In media containing ampicillin (25 to 100 mg/liter), daptomycin MICs decreased from 1.0 to 0.38 mg/liter. Based on time-kill analysis and an in vitro pharmacodynamic model, ampicillin enhanced daptomycin activity against the study VRE from a bacteriostatic to a bactericidal profile. VRE grown in ampicillin (25 to 150 mg/liter) demonstrated an incremental reduction in its relative net positive surface charge. When grown in the presence (versus absence) of ampicillin (25 and 100 mg/liter), the VRE strain (i) was more susceptible to killing by LL-37, tPMPs, hNP-1, and RP-1 but not to polymyxin B and (ii) exhibited greater binding to Bodipy-labeled daptomycin. We conclude that ampicillin induces reductions in net positive bacterial surface charge of VRE, correlating with enhanced bactericidal effects of cationic calcium-daptomycin and a diverse range of other cationic peptides in vitro. While the mechanism(s) of such β-lactam-mediated shifts in surface charge remains to be defined, these finding suggest a potential for β-lactam-mediated enhancement of activity of both daptomycin and innate host defense peptides against antibiotic-resistant bacteria.

Double-shell gold nanoparticle-based DNA-carriers with poly- l-lysine binding surface

In view of the prospective applications of polyamine coatings in functional gold nanoparticles for use as carriers in gene delivery systems, in tissue repair and as bactericidal and virucidal non-toxic vehicle, we have investigated the interactions of poly- l-lysine (PLL) with gold nanoparticles (AuNP). Since direct binding of PLL to AuNP is not strong at neutral pH, we have focused on PLL interactions with carboxylated self-assembled monolayers (SAM) on AuNP, such as the citrate-capped AuNP. The double-shell nanoparticles AuNP@Cit/PLL thus produced do not contain any toxic thiols. We have observed strong electrostatic interactions between polycationic chains of PLL and AuNP@Cit in weakly acidic to weakly alkaline solutions (pH 5–9), as evidenced by the bathochromic shift of the local surface plasmon (SP) band and strong increase in resonance elastic light scattering (RELS) intensity. The stoichiometry of interactions evaluated on the basis of RELS data indicates on a hyper-Langmuirian type of interactions with stoichiometric coefficient n = 1.35 (PLL : AuNP@Cit). From the RELS titration data, a shift of the deprotonation constant for the bound PLL has been determined ( pK a = 11.6 for the bound PLL vs. 10.48 for the free PLL). The deprotonation of PLL leads to AuNP aggregate disassembly, evidenced by sharp RELS decline and hypsochromic shift of SP band. We have found that under these conditions, a residual aggregation due to the interparticle interactions between β-sheets of PLL overcoat become predominant. The molecular dynamics simulations indicate that multiple hydrogen bonds can also be formed between the PLL linker and the shell molecules of AuNP@Cit. The double-shell nanoparticles, AuNP@Cit/PLL, have been shown to attract DNA molecules using highly sensitive RELS measurements presenting the proof-of-concept for the suitability of this non-toxic nanostructured material for gene delivery applications. The advantage of the proposed material is no toxicity related to the ligand release in gene delivery processes in contrast to the thiol-functionalized AuNP.

L-lysine coated iron oxide nanoparticles: Synthesis, structural and conductivity characterization

l-lysine coated iron oxide (LCIO) nanoparticles were synthesized by a co-precipitation method in the presence of amino acid. XRD analysis confirmed the presence of cubic magnetite phase with an average crystallite size of 8 ± 4 nm. Particle size estimated from TEM, by log-normal fitting, is ∼114 nm. The difference between the crystallite size from XRD and particle size from TEM indicates polycrystalline nature of synthesized particles. FT-IR show that the binding of l-lysine on the surface of iron oxide through carboxyl groups is via unidentate linkage. The presence of l-lysine on iron oxide is also confirmed by zeta potential measurements on LCIO nanoparticles, revealing a partial coverage of iron oxide with l-lysine. In order to obtain chemically stable, well-dispersed and uniform sized nanoparticles, amino acids are suitable because they play a very important role in the body. Conductivity measurements were performed to investigate the influence of the coating on the conduction characteristics of iron oxide and results show the existence of a hopping conduction mechanism. Magnetic transition is observed at ∼70 °C for uncoated iron oxide and LCIO samples. Frequency (1 Hz to 3 MHz) and temperature (290–420 K) dependant AC conductivity measurements have resulted in AC activation energies between 0.048 and 0.041 eV for uncoated and 0.050–0.044 eV for LCIO nanoparticles. Temperature-dependant DC resistivity measurements of iron oxide and LCIO at high temperatures resulted in the DC activation energies of 0.22 and 0.43 eV respectively. The higher activation energy value for LCIO is the result of coating by insulating l-lysine layer.

Dipeptidyl peptidase IV (DPP IV/CD26) is a cell-surface plasminogen receptor

Binding of plasminogen (Pg) to cell-surface receptors colocalized with plasminogen activators promotes Pg activation and enables cells to utilize the proteolytic activity of plasmin (Pm). Proteolysis by Pm is necessary in several physiological and pathological processes requiring extracellular matrix degradation including cell migration, tumor cell invasion and metastasis. The binding of Pg to cell-surface receptors is regulated by two major structural features: L-lysine binding sites (LBS) and negatively charged sialic acid residues located on its carbohydrate chains. Pg uses its LBS to bind to a wide spectrum of cell-surface receptors whereas binding through its sialic acid residues is limited only to receptor proteins containing cationic pockets or lectin-like modules. In this review, we discuss both mechanisms, including the identification of DPP IV as a Pg receptor and the possible physiological role of Pg/Pm in complex with DPP IV and adenosine deaminase (ADA) and /or the Na+/H+ exchanger isoform NHE-3 in prostate cancer.

Binding Energy in the One-Electron Reductive Cleavage of S-Adenosylmethionine in Lysine 2,3-Aminomutase, a Radical SAM Enzyme

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes. The energetics is here reported for the action of lysine 2,3-aminomutase (LAM), which catalyzes the interconversion of l-lysine and l-beta-lysine. From earlier work, the reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the cluster (Hinckley, G. T., and Frey, P. A. (2006) Biochemistry 45, 3219-3225), 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding l-lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and l-alanine in place of l-lysine, with S-adenosyl-l-homocysteine (SAH) bound to the cluster in the presence of l-lysine, and with SAH bound to the cluster in the presence of l-alanine or of l-alanine and ethylamine in place of l-lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosyl-l-methionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of l-lysine to LAM contributes 4 kcal mol-1, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal mol-1 toward lowering the barrier for reductive cleavage of SAM from 32 kcal mol-1 in solution to 9 kcal mol-1 at the active site of LAM.

Discrimination of cognate and noncognate substrates at the active site of class II lysyl-tRNA synthetase.

Within the two unrelated aminoacyl-tRNA synthetase classes, lysyl-tRNA synthetase (LysRS) is the only example known to exist in both classes. To probe the role of the amino acids responsible for L-lysine binding in the active site of the class II LysRS (LysRS2), we studied the lysS-encoded Escherichia coli protein. On the basis of the structure of L-lysine complexed with E. coli LysRS2 (lysS), residues implicated in amino acid recognition and discrimination were systematically replaced. Steady-state kinetic parameters for these variants showed reductions in the catalytic efficiency (k(cat)/K(M)) of 1-3 orders of magnitude, allowing the assignment of specific roles for key residues in the active site of LysRS2. To further investigate the role of each residue in discrimination against noncognate amino acids, steady-state kinetic parameters were determined for the nonprotein amino acid S-(2-aminoethyl)-L-cysteine, a potent inhibitor of LysRS2. While a number of variants showed reductions of several hundred-fold in efficiency of S-(2-aminoethyl)-L-cysteine utilization, this was uniformly accompanied by similar reductions in the efficiency of lysine utilization. Thus, manipulation of the amino acid binding site only allowed up to a 4-fold improvement in S-(2-aminoethyl)-L-cysteine discrimination. This is in contrast to the highly effective discrimination against S-(2-aminoethyl)-L-cysteine by class I LysRS and correlates with the fundamentally different roles of conserved aromatic residues in the two LysRS active sites. This now provides a mechanistic basis for the proposal that differences in amino acid discrimination have been pivotal in the evolution of two unrelated LysRSs.

Characterization of the L-lysine biosynthetic pathway in the obligate methylotroph Methylophilus methylotrophus.

The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.

L-Lysine acts like a partial serotonin receptor 4 antagonist and inhibits serotonin-mediated intestinal pathologies and anxiety in rats.

The purpose of this investigation was to determine whether a nutritionally essential amino acid, l-lysine, acts like a serotonin receptor 4 (5-HT4) antagonist, and if l-lysine is beneficial in animal models of serotonin (5-HT)-induced anxiety, diarrhea, ileum contractions, and tachycardia and in stress-induced fecal excretion. The radioligand-binding assay was used to test the binding of l-lysine to various 5-HT receptors. The effects of l-lysine on 5-HT-induced contractions of isolated guinea pig ileum were studied in vitro. The effects of oral administration of l-lysine on diarrhea, stress-induced fecal excretion, and 5-HT-induced corticosterone release, tachycardia, and anxiety (an elevated plus maze paradigm) were studied in rats in vivo. l-Lysine (0.8 mmol/dl) inhibited (9.17%) binding of 5-HT to the 5-HT4 receptor, without any effect on 5-HT1A,2A,2B,2C,3 binding. l-Lysine (0.07 and 0.7 mmol/dl) blocked 5-HT-induced contractions of an isolated guinea pig ileum in vitro (P < 0.05 and P < 0.01). Orally applied l-lysine (1 g/kg of body weight) inhibited (P < 0.12) diarrhea triggered by coadministration of restraint stress and 5-hydroxytryptophane (10 mg/kg of body weight), and significantly blocked anxiety induced by the 5-HT4 receptor agonist (3.0 mmol/liter) in rats in vivo. No effects of l-lysine or the 5-HT4 receptor agonist on plasma corticosterone and heart rate were recorded. l-Lysine may be a partial 5-HT4 receptor antagonist and suppresses 5-HT4 receptor-mediated intestinal pathologies and anxiety in rats. An increase in nutritional load of l-lysine might be a useful tool in treating stress-induced anxiety and 5-HT-related diarrhea-type intestinal dysfunctions.

Lysyl-tRNA Synthetase from Bacillus stearothermophilus: The Trp314 Residue is Shielded in a Non-polar Environment and is Responsible for the Fluorescence Changes Observed in the Amino Acid Activation Reaction

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of l-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess l-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20Å away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the l-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of l-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the l-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.

Tissue factor is the receptor for plasminogen type 1 on 1-LN human prostate cancer cells

Tissue factor (TF), the initiator of the extrinsic pathway of coagulation, binds plasminogen (Pg) with high affinity through an interaction between kringles 1-3 of Pg and the extracellular domain of TF. We investigated the binding of Pg type 1 (Pg 1) and Pg type 2 (Pg 2) to highly invasive, TF-expressing, 1-LN human prostate tumor cells and to TF isolated from 1-LN cell membranes. Pg 1, containing both N-linked and O-linked oligosaccharide chains, bound to isolated TF with high affinity, whereas Pg 2, containing only one O-linked oligosaccharide chain, did not bind to TF. Although Pg 1 and Pg 2 bind to 1-LN cells, only anti-TF antibodies inhibited the binding of Pg 1, suggesting that TF functions as the receptor for Pg 1 on 1-LN cells. Binding of Pg 1 to isolated TF was inhibited by 6-aminohexanoic acid and alpha-methylmannoside, suggesting that Pg 1 L-lysine binding sites and the biantennary, mannose-containing N-linked oligosaccharide chain are involved in this interaction. Binding of Pg 1 to 1-LN cells promoted activation by receptor-bound urinary-type Pg activator (u-PA) and initiated a Ca(++) signaling cascade. In previous studies we demonstrated that the Pg 2 O-linked carbohydrate chain is essential for its binding to CD26 on 1-LN cells. The current studies suggest that Pg oligosaccharide chains regulate the binding of Pg 1 and Pg 2 to separate receptors on the cell surface.

1D1715 B.stearothermophilus由来リジンtRNA合成酵素のL-リジン結合部位に存在する芳香族アミノ酸残基の関与

1D1715 B.stearothermophilus由来リジンtRNA合成酵素のL-リジン結合部位に存在する芳香族アミノ酸残基の関与

Lysyl-tRNA synthetase from Bacillus stearothermophilus. Stopped-flow kinetic analysis of enzyme.lysyladenylate formation.

Amino acid activation reaction of the lysyl-tRNA synthetase [L-lysine:tRNALys ligase (AMP forming); EC 6.1.1.6] from Bacillus stearothermophilus was studied fluorometrically by the stopped-flow method. The addition of L-lysine to the enzyme solution caused quenching of the protein fluorescence and the subsequent addition of ATP restored the quenched fluorescence [Takita et al. (1996) J. Biochem. 119, 680-689; Takita et al. (1997) 121, 244-250]. In the stopped-flow analysis, however, the former fluorescence change (quenching) could not be detected, while the latter change (restoration) was detectable. The L-lysine binding process was suggested to be much faster than the ATP binding process, being completed within the dead-time of the apparatus, ca. 3 ms. The hyperbolic dependence of kapp on the initial ATP concentration suggested that the ATP binding to the enzyme.L-lysine complex followed a two-step mechanism. Two L-lysine analogues that exhibit the qualitatively similar behavior to L-lysine in the fluorometric titration, L-lysine hydroxamate and L-lysine amide, were examined similarly. The two-step process was also suggested for these analogues, and the forward rate constant in the rate-determining step for L-lysine amide (221+/-7 s-1) was significantly larger than those for L-lysine (45.7+/-4.6 s-1) and L-lysine hydroxamate (14. 5+/-1.7 s-1) at pH 8.0, 30 degrees C.

L-arginine binding to liver arginase requires proton transfer to gateway residue His141 and coordination of the guanidinium group to the dimanganese(II,II) center.

Rat liver arginase contains a dimanganese(II,II) center per subunit that is required for catalytic hydrolysis of l-arginine to form urea and l-ornithine. A recent crystallographic study has shown that the Mn2 center consists of two coordinatively inequivalent manganese(II) ions, MnA and MnB, bridged by a water (hydroxide) molecule and two aspartate residues [Kanyo et al. (1996) Nature 383, 554-557]. A conserved residue, His141, is located near the proposed substrate binding region at 4.2 A from the bridging solvent molecule. The present EPR studies reveal that there is no essential alteration of the Mn2 site upon mutation of His141 to an Asn residue, which lacks a potential acid/base residue, while the catalytic activity of the mutant enzyme is 10 times lower vs wild-type enzyme. The binding affinity of l-lysine, l-arginine (substrate), and Nomega-OH-l-arginine (type 2 binders) increases inversely with the pKa of the side chain. Binding of l-lysine is more than 10 times weaker, and the substrate Michaelis constant (Km) is >6-fold greater (weaker binding) in the His141Asn mutant than in wild-type arginase. L-Lysine and Nomega-OH-L-arginine, type 2 binders, induce extensive loss of the EPR intensity, suggesting direct coordination to the Mn2 center. From these data and the pH dependence of type 2 binders, we conclude that His141 functions as the base for deprotonation of the side-chain amino group of L-lysine and the substrate guanidinium group, -NH-C(NH2)2+ and that the unprotonated side chain of these amino acids is responsible for binding to the active site. A different class of inhibitors (type 1), including L-isoleucine, L-ornithine, and L-citrulline, suppresses enzymatic activity, producing only minor change in the zero-field splitting of the Mn2 EPR signal and no change in the EPR intensity, suggestive of minimal conformational transformation. We propose that type 1 alpha-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the alpha-aminocarboxylate groups of the substrate. A new mechanism for the arginase-catalyzed hydrolysis of L-arginine is proposed which has general relevance to all binuclear hydrolases: (1) Deprotonation of substrate l-arginine(H+) by His141 permits entry of the neutral guanidinium group into the buried Mn2 region. Binding of the substrate imino group (>C=NH), most likely to MnB, is coupled to breaking of the MnB-(mu-H2O) bond, forming a terminal aquo ligand on MnA. (2) Proton transfer from the terminal MnA-aqua ligand to the substrate Ndelta-guanidino atom forms the nucleophilic hydroxide on MnA and the cationic NdeltaH2+-guanidino leaving group. Protonation of the substrate -NdeltaH2+-group is likely assisted by hydrogen bonding to the juxtaposed anionic carboxylate group of Glu277. (3) Attack of the MnA-bound hydroxide at the electrophilic guanidino C-atom forms a tetrahedral intermediate. (4) Formation of products is initiated by cleavage of the Cepsilon-NdeltaH2+ bond, yielding urea and L-ornithine(H+).

Proton NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli [Cleary et al. (1989) Biochemistry 28, 1884-1891]. The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3. Thus, the His48a and His64 side chains are in solvent-shielded locations. As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)

L-[3H]lysine binding to rat retinal membrane: I. Quantitative determination and characterization of the binding sites.

A saturable reversible binding to membranes from rat retina has been found for L-[3H]lysine. Specific binding is time, temperature and protein concentration-dependent, and shows stereospecificity. The best computer fits of the experimental data are obtained with a receptor model based on two independent binding sites, of which only one site with a Kd value of 229.4 +/- 14.23 nM and a Bmax of 2.04 +/- 0.11 pmol/mg protein could be characterized satisfactorily. Several compounds included putative neurotransmitters have moderate or no affinity for L-lysine binding sites. A different pattern of distribution of L-[3H]lysine binding sites is observed among various regions of the brain, with the highest density in the occipital cortex, and the lowest density in ponsmedulla. The existence of binding sites in rat retinal membranes for L-lysine, as well as in the areas involved in the visual pathway, suggests a role for this amino acid in the physiological mechanism of the visual function.

Cross-Linking of IgE-Receptor complexes at the cell surface: A fluorescence method for studying the binding of monovalent and bivalent haptens to IgE

We have developed a method for use in investigating factors controlling the binding and cross-linking by bivalent haptens of immunoglobulin E (IgE) bound to receptors on rat basophilic leukemia (RBL) cells. This method employs monoclonal anti-2,4-dinitrophenyl (DNP) IgE that is labeled with fluorescein-5-isothiocyanate (FITC), and it measures FITC quenching that accompanies DNP occupation of the antibody combining sites in a titration experiment. The validity of this approach is demonstrated using the monovalent hapten DNP- l-lysine. The affinity constant for this ligand obtained by the FITC quenching method is compared with those obtained with previously established methods: equilibrium dialysis and quenching of endogenous tryptophan for IgE in solution and [ 3H]-DNP- l-lysine binding to IgE on cells. The FITC quenching method has been used to carry out a detailed study of the binding of monovalent DNP-aminocapryol l-tyrosine (DCT) and bivalent (DCT) 2-cystine to FITC-IgE and its Fab fragments in solution. Intrinsic ( K) and cross-linking ( K x) affinity constants are obtained by analyzing the binding curves in terms of simple equilibrium equations. With these DCT haptens the ability of this method to assess hapten binding and cross-linking of IgE bound to receptors on RBL cells is shown.

Transport of L-lysine by rat renal brush border membrane vesicles.

L-3H-lysine uptake into brush border membrane vesicles was measured by a rapid filtration technique. A significant binding of L-lysine at the vesicle interior was observed. Extrapolating initial linear uptake to zero incubation time did not indicate binding of the amino acid to the external membrane surface. Sodium stimulated the L-lysine uptake specifically. Experiments in the presence of potassium/valinomycin induced diffusion potentials, and experiments with a potential sensitive fluorescent dye documented an electrogenic uptake mechanism for L-lysine only in the presence of sodium. Sodium independent uptake proceeds via an electroneutral pathway. Transstimulation experiments show carrier mediated uptake in the presence and absence of sodium. An outwardly directed proton-gradient stimulated L-lysine uptake in the presence and absence of sodium. Saturation of L-lysine uptake was observed in the presence and absence of sodium. In the absence of sodium, L-lysine uptake was inhibited by L-arginine, L-cystine, L-phenylalanine and L-methionine. The sodium dependent uptake was inhibited by L-arginine and L-cysteine; small inhibition by L-phenylalanine was observed. In the presence or absence of sodium, L-lysine uptake was inhibited neither by D-lysine nor by L-glutamic acid. These results document carrier mediated transport of L-lysine via (a) transport mechanism(s) not obligatory requiring sodium.