Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.     

The iron-binding properties of aminochelin, the mono(catecholamide) siderophore of Azotobacter vinelandii

Azotobacter vinelandii produces siderophores with different metal-binding properties, depending on the concentration of Fe(III) and molybdate in the growth medium. The three protonation constants of the mono(catecholamide) siderophore aminochelin were determined by simultaneous spectrophotometric and potentiometric titrations as log K(1)=12.1, log K(2)=10.22 and log K(3)=7.04. Based on the two catechol protonation constants, log K(1) and log K(3), the overall stability constant of the aminochelin iron 3:1 complex was found to be log beta(3)=41.3, resulting in a pFe(3+) value of 17.6 at pH 7.45. In order to further investigate the properties of the siderophore, the solubilization of Fe(III) hydroxide by a 8x10(-4) M solution of aminochelin at pH 7 and 25 degrees C was followed spectrophotometrically in the absence and in the presence of molybdate. It was observed that the addition of molybdate resulted in a significant delay in the solubilization.     

Cofactor-type inhibitors of inosine monophosphate dehydrogenase via modular approach: targeting the pyrophosphate binding sub-domain

Cofactor-type inhibitors of inosine monophosphate dehydrogenase (IMPDH) that target the nicotinamide adenine dinucleotide (NAD) binding domain of the enzyme are modular in nature. They interact with the three sub-sites of the cofactor binding domain; the nicotinamide monophosphate (NMN) binding sub-site (N sub-site), the adenosine monophosphate (AMP) binding sub-site (A sub-site), and the pyrophosphate binding sub-site (P sub-site or P-groove). Mycophenolic acid (MPA) shows high affinity to the N sub-site of human IMPDH mimicking NMN binding. We found that the attachment of adenosine to the MPA through variety of linkers afforded numerous mycophenolic adenine dinucleotide (MAD) analogues that inhibit the two isoforms of the human enzyme in low nanomolar to low micromolar range. An analogue 4, in which 2-ethyladenosine is attached to the mycophenolic alcohol moiety through the difluoromethylenebis(phosphonate) linker, was found to be a potent inhibitor of hIMPDH1 (K(i)=5 nM), and one of the most potent, sub-micromolar inhibitor of leukemia K562 cells proliferation (IC(50)=0.45 μM). Compound 4 was as potent as Gleevec (IC(50)=0.56 μM) heralded as a 'magic bullet' against chronic myelogenous leukemia (CML). MAD analogues 7 and 8 containing an extended ethylenebis(phosphonate) linkage showed low nanomolar inhibition of IMPDH and low micromolar inhibition of K562 cells proliferation. Some novel MAD analogues described herein containing linkers of different length and geometry were found to inhibit IMPDH with K(i)'s lower than 100 nM. Thus, such linkers can be used for connection of other molecular fragments with high affinity to the N- and A-sub-site of IMPDH.     

Modeling the haloperoxidases: reversible oxygen atom transfer between bromide ion and an oxo-Mn(V) porphyrin

The manganese meso-dimethylimidazolium porphyrin complex Mn(III)[TDMImP] reacted with HOBr/OBr(-) to generate the corresponding oxo-Mn(V)[TDMImP] species. The rate of this process accelerated with increasing pH. A forward rate constant, k(for), of 1.65x10(6)M(-1)s(-1) was determined at pH 8. Under these conditions, the oxo-Mn(V) species is short-lived and is transformed into the corresponding oxo-Mn(IV) complex. A first-order rate constant, k(obs), of 0.66 s(-1) was found for this reduction process at pH 8. The mechanism of this reduction process, which was dependent on bromide ion, appeared to proceed via an intermediate Mn(III)-O-Br complex. Thus, both a fast, reversible Mn(III)-O-Br bond heterolysis and a slower homolytic pathway occur in parallel in this system. The reverse oxidation reaction between oxo-Mn(V)[TDMImP] and bromide was investigated as a function of pH. The rate of this oxo-transfer reaction (k(rev)=1.4x10(3)M(-1)s(-1) at pH 8) markedly accelerated as the pH was lowered. The observed first-order dependence of the rate on [H(+)] indicates that the reactive species responsible for bromide oxidation is a protonated oxo-hydroxo complex and the stable species present in solution at high pH is dioxo-Mn(V)[TDMImP], [O=Mn(V)=O](-). The oxo-Mn(V) species retains nearly all of the oxidative driving force of the hypohalite. The equilibrium constant K(equi)=k(for)/k(rev) for the reversible process was determined at three different pH values (K(equi)=1.15x10(3) at pH 8) allowing the measurement of the redox potentials E of oxo-Mn(V)/Mn(III) (E=1.01 V at pH 8). The redox potential for this couple was extrapolated over the entire pH scale using the Nernst relationship and compared to those of the manganese 2- and 4-meso-N-methylpyridinium porphyrin couples oxo-Mn(V)[2-TMPyP]/Mn(III)[2-TMPyP], oxo-Mn(V)[4-TMPyP]/Mn(III)[4-TMPyP], OBr(-)/Br(-) and H(2)O(2)/H(2)O. Notably, the redox potential of oxo-Mn(V)/Mn(III) for the imidazolium porphyrin approaches that of H(2)O(2)/H(2)O at low pH.     

Dynamics of pH-dependent self-association and membrane binding of a dicarboxylic porphyrin: a study with small unilamellar vesicles

Steady-state and stopped-flow measurements of the absorbance and fluorescence of aqueous solutions were performed to characterize the pH-dependent ionization and aggregation states of deuteroporphyrin. Porphyrin self-association promoted by neutralization of the carboxylic groups takes place within a few milliseconds impeding characterization of the monomer ionization states. Extrapolation at infinite dilution of the values obtained from steady-state measurements yielded the pKs of the carboxylic groups (6.6, 5.3) and inner nitrogens (4.1, 2.3). The kinetics of interactions of the porphyrin with unilamellar fluid state dioleoylphosphatidylcholine vesicles was examined in a large pH range, with focus on the entry step. From alkaline pH to a value of 6.5, the entrance rate is maximal (1.69 x 10(6) M(-1) s(-1) versus phospholipid concentration). It decreases to 2.07 x 10(5) M(-1) s(-1) at lower pH with an apparent pK of 5.39. This effect appears to be related to the formation of porphyrin dimer rather than to the protonation of inner nitrogen. In keeping with previous data, these results support the concept of a pH-mediated selectivity of carboxylic porphyrins for tumor. They also indicate that the propensity of these molecules to self-associate at low pH could yield to some retention in acidic intracellular vesicles of the endosome/lysosome compartment.     

Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition

The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent results: Michaelis constant K(m)=3.3mM, catalytic constant k(cat)=1.7 x 10(3)s(-1), bimolecular rate constant k(s)=5.1 x 10(5)M(-1)s(-1). The reaction product, choline, was found to be a competitive inhibitor with a dissociation constant K(i)=9.1mM. Betaine had a slightly higher affinity for the enzyme, but the inhibition was only partial. This study confirms the usefulness of microcalorimetry for the kinetic study of enzymes and their inhibitors.     

The molybdate binding protein Mop from Haemophilus influenzae--biochemical and thermodynamic characterisation

The protein Mop from Haemophilus influenzae is a member of the molbindin family of proteins. Using isothermal titration calorimetry (ITC), Mop was observed to bind molybdate at two distinct sites with a stoichiometry of 8 mol molybdate per Mop hexamer. Six moles of molybdate bound endothermically at high affinity sites (K(a)=8.5 x 10(7)M(-1)), while 2 mol of molybdate bound exothermically at lower affinity sites (K(a)=3.7 x 10(7)M(-1)). Sulphate was also found to bind weakly at the higher affinity sites. ITC revealed that the affinity of molybdate binding to the endothermic site decreased with increasing pH and was accompanied by the transfer from the buffer to the protein of one proton per Mop monomer. These kinetic and thermodynamic results are interpreted with reference to molbindin crystal structures and data concerning molbindin binding affinities. Mop binds molybdate with high specificity, capacity, and affinity which indicates that Mop has a role as an intracellular molybdate binding protein involved in oxyanion homeostasis.     

Characterization of a metalloregulatory bismuth(III) site in Staphylococcus aureus pI258 CadC repressor

Staphylococcus aureus pI258 CadC is a metal sensor protein that regulates the expression of the cad operon which encodes metal ion resistance proteins involved in the efficient efflux of Cd(II), Pb(II), Zn(II) and, according to one report, Bi(III) ions. In this paper, direct evidence is presented that Bi(III) binds to CadC and negatively regulates cad operator/promoter (O/P) binding. Optical absorption spectroscopy reveals that dimeric CadC binds approximately 0.8 mol equivalents of Bi(III) per CadC monomer to form a coordination complex characterized by three S(-)-->Bi(III) ligand-to-metal charge transfer transitions, with the longest wavelength absorption band centered at 415 nm (epsilon(415)=4000 M(Bi)(-1) cm(-1)). UV-Vis absorption spectra of wild-type and mutant Cys-->Gly (Ser) substitution CadC mutants compared to [Bi(DTT)(2)], [Bi(GSH)(3)] and [Bi(NAC)](3) model complexes reveal that Cys7, Cys11, Cys60 and Cys58 directly coordinate Bi(III) in a tetrathiolate coordination complex. The apparent affinity derived from a Bi(III)-displacement optical titration with Cd(II) is estimated to be K(Bi)< or =10(12) M(-1). Apo-CadC binds with high affinity [ K(a)=1.1(+/-0.3)x10(9) M(-1); 0.40 M NaCl, pH 7.0, 25 degrees C] to a 5'-fluorescein-labeled cad O/P oligonucleotide,while the binding of one molar equivalent of Bi(III) per CadC monomer (Bi(1)-CadC) reduces the affinity by approximately 170-fold. Strikingly, Bi(III)-responsive negative regulation of cad O/P binding is abrogated for Bi(1)-C60G CadC and severely disrupted in Bi(1)-C7G CadC, whose relative affinity is reduced only 10-fold. The mechanism of Bi(III)-responsive metalloregulation is discussed, based on the findings presented here. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0336-9.     

High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K(d) < 10(-)(9) M) and slow dissociation rates (t(1/2) > 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K(d) = 1.2 x 10(-)(7) M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by 相似文献   

Reductive nitrosylation of water-soluble iron porphyrins by S-nitroso-N-acetylpenicillamine: rate constants and EPR characterization

Reductive nitrosylation of the water-soluble iron derivatives of the cationic Fe(III)(TMPyP) and anionic Fe(III)(TPPS) porphyrins [where TMPyP=tetra-meso-(4-N-methylpyridiniumyl)porphinate and TPPS=tetra-meso-(4-sulfonatophenyl)porphinate] by the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) was studied using optical absorption spectroscopy and electron paramagnetic resonance. Nitrosylation rates were obtained, the reaction was found to be first order in the SNAP concentration and the stoichiometry of the reaction was one to one. The similarity between the obtained second-order rate constants for both porphyrins, k(TMPyP)=0.84 x 10(3)M(-1)s(-1) and k(TPPS)=0.97 x 10(3)M(-1)s(-1), suggested that the reaction mechanism is approximately independent of the nature of the porphyrin meso-substituents. A mechanism was proposed involving the hydrolysis of SNAP by an out of plane liganded H(2)O yielding the sulfenic acid of N-acetylpenicillamine and the transfer of NO(-) to Fe(III). The EPR (electron paramagnetic resonance) spectra of the SNAP- and gaseous NO-treated porphyrins were obtained and compared. The difference between the spectra of the cationic and anionic porphyrins indicates different local symmetry and Fe-N-O bond angle. SNAP-treatment produced much more resolved hyperfine structures than gaseous NO-treatment.     

Recombinant soluble human Fcgamma receptor I with picomolar affinity for immunoglobulin G

The ectodomain of human FcgammaRI (rsCD64) was expressed in HEK 293T cells and purified by immobilized-metal affinity chromatography. Binding activity to human IgG was verified by ELISA and the isotype-specificity determined by a surface plasmon resonance inhibition assay was found to be the same as for native CD64. The active concentration of the rsCD64 preparation was derived using a solution competition assay and was used for the subsequent kinetic analysis. Binding curves were well described by a simple monovalent interaction model confirming the known stoichiometry of the interaction. Mass-transport limitation was prevented by using sufficiently low surface capacities. For binding to the recombinant mouse/human chimeric antibody cPIPP (IgG1/kappa) a high association rate of k(ass)=1.7 x 10(6) (M s)(-1) and a low dissociation rate of k(diss)=1.8 x 10(-4) s(-1) were observed. The derived dissociation equilibrium constant of K(D)=110 pM was significantly lower than that reported for binding to native FcgammaRI.     

Spectrofluorimetric study of porphyrin incorporation into membrane models--evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.     

Efficient photodynamic therapy of cancer using chemotherapeutic porphyrin-ruthenium metalla-cubes

Two cationic octanuclear metalla-cubes [Ru(8)(η(6)-C(6)H(5)Me)(8)(tpp-H2)(2)(dhbq)(4)](8+) and [Ru(8)(η(6)-p-iPrC(6)H(4)Me)(8)(tpp-H2)(2)(dhbq)(4)](8+) were prepared and evaluated as dual photosensitizers and chemotherapeutics in cancer cells. In the dark, the complexes presented high cytotoxicity towards only melanoma and ovarian cancer cells. However, the complexes exhibited good phototoxicities toward all cancer cells (1μM concentration, LD(50)=2-7J/cm(2)), thus suggesting a dual synergistic effect with good properties of both the arene ruthenium chemotherapeutics and the porphyrin photosensitizers.     

Kinetics behaviors of Na,K-ATPase: comparison of solubilized and DPPC:DPPE-liposome reconstituted enzyme

We describe and compare the main kinetic characteristics of rabbit kidney Na,K-ATPase incorporated inside-out in DPPC:DPPE-liposomes with the C(12)E(8) solubilized and purified form. In proteoliposomes, we observed that the ATP hydrolysis of the enzyme is favored and also its affinity for Na(+)-binding sites increases, keeping the negative cooperativity with two classes of hydrolysis sites: one of high affinity (K(0.5)=6 microM and 4 microM for reconstituted enzyme and purified form, respectively) and another of low affinity (K(0.5)=0.4 mM and 1.4 mM for reconstituted enzyme and purified form, respectively). Our data showed a biphasic curve for ATP hydrolysis, suggesting the presence of (alphabeta)(2) oligomer in reconstituted Na,K-ATPase similar to the solubilized enzyme. The Mg(2+) concentration dependence in the proteoliposomes stimulated the Na,K-ATPase activity up to 476 U/mg with a K(0.5) value of 0.4 mM. The Na(+) ions also presented a single saturation curve with V(M)=551 U/mg and K(0.5)=0.2 mM with cooperative effects. The activity was also stimulated by K(+) ions through a single curve of saturation sites (K(0.5)=2.8 mM), with cooperative effects and V(M)=641 U/mg. The lipid microenvironment close to the proteic structure and the K(+) internal to the liposome has a key role in enzyme regulation, affecting its kinetic parameters while it can also modulate the enzyme's affinity for substrate and ions.     

beta-1,3-Glucan binding by a thermostable carbohydrate-binding module from Thermotoga maritima.

The C-terminal 155 amino acids of the putative laminarinase, Lam16A, from T. maritima comprise a highly thermostable family 4 CBM that binds beta-1,3- and beta-(1,3)(1,4)-glucans. Laminarin, a beta-1,3-glucan, presented two classes of binding sites for TmCBM4-2, one with a very high affinity (3.5 x 10(7) M(-1)) and one with a 100-fold lower affinity (2.4 x 10(5) M(-1)). The affinities for laminarioligosaccharides and beta-(1,3)(1,4)-glucans ranged from approximately 2 x 10(5) to approximately 2.5 x 10(6) M(-1). Cellooligosaccharides and laminariobiose were bound only very weakly (K(a)s approximately 5 x 10(3) M(-1)). Spectroscopic and mutagenic studies implicated the involvement of three tryptophan residues (W28, W58, and W99) and one tyrosine residue (Y23) in ligand binding. Binding was enthalpically driven and associated with large negative changes in heat capacity. Temperature and osmotic conditions profoundly influenced binding. For the first time in solution, the direct uptake and release of water in CBM binding are demonstrated.     

Antiferritin VL homodimer binds human spleen ferritin with high specificity

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.     

Specific interactions between cryogel components: role of extra domain A containing fibronectin in cryogelation

Cryogel is a physical gel formed by heterophilic aggregation of extra domain A containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep), which are found in high concentrations in the blood of patients suffering from rheumatoid arthritis. In this study, we clarify the specific interactions between cryogel components in terms of the affinity constant (K(A)), obtained by surface plasmon resonance (SPR). It is found that Fbg self-interactions occur at lower temperatures, and that K(A) of Fbg-Hep changes with temperature. Specifically, K(A) (2.0 x 10(8) [M(-1)]) of Fbg-Hep at 5 degrees C increases significantly from that (1.0x10(7) [M(-1)]) at 40 degrees C. K(A) of EDA(+)FN-Hep increases with temperature, by approximately 100-fold between 40 degrees C (K(A)=10(12) [M(-1)]) and 20 degrees C (K(A)=10(10) [M(-1)]). Although K(A) of the FN fragments of Hep-binding domain containing an EDA region [EDA(+)HBD(+)] and Hep increases with temperatures above 30 degrees C, K(A)s of HBD(+)-Hep and EDA(+)-Hep are not temperature-dependent. Therefore, EDA(+)HBD(+), formed as a special structure for high Hep affinity, exhibits temperature-dependent interaction with Hep. These results suggest that the main role of EDA(+)FN in cryogelation is to support the interaction with Hep.     

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.     

Converting weak binders into infinite binders

Monoclonal antibody 2D12.5 binds DOTA chelates of all the rare earths with K(d) approximately 10(-)(8) M, making it useful for the capture of probe molecules with a variety of properties. To make 2D12.5 even more useful for biological applications, we have engineered a single cysteine residue at position 54 of the heavy chain, a site proximal to the protein's binding site, so that weakly electrophilic metal complexes of (S)-2-(4-acrylamidobenzyl)-DOTA (AABD) may bind and form permanent linkages. At 37 degrees C, pH 7.5, all of the rare earth-AABD complexes bind permanently to the 2D12.5 G54C mutant within 5 min, in yields that correlate with their relative binding affinities. Surprisingly, indium-AABD also binds permanently in >50% yield within 5 min, despite the fact that changing the metal to indium reduces the affinity approximately 100x; even copper-AABD, which has approximately 10 000x lower binding affinity than the rare earths, binds permanently in >70% yield within 2 h. However, acrylamido compounds with no measurable affinity do not bind permanently. The important practical implication is that the G54C mutant of 2D12.5 may be used for applications that include not only the rare earths, but also an unexpected range of other elements as well. This infinite binding system can exhibit selective and permanent attachment with a remarkable range of structurally related ligands, albeit at slower rates as affinities decrease.     

Specificity of the synergistic anion for iron binding by ferric binding protein from Neisseria gonorrhoeae

Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.