Characterization and metal affinity of Tirofiban, a pharmaceutical compound used in acute coronary syndromes

The crystal and molecular structure of Tirofiban [N-(n-butanesulfonyl)-O-(4-(4-piperidinyl)-butyl)-(S)-tyrosine] is here reported. In the solid state the carboxylic group is in the anionic form while the piperidine molecule appear in the protonated form. By H NMR spectroscopy and potentiometric study three pKa are found: pKa(COOH) = 3.1 (1), pKa(NHPIP) = 11.6(1) and pKa(NHSO2) = 13.8(1). The complexing ability of Tirofiban towards various metal ions (Cu(II), Ni(II), Co(II), Cd(II), Pb(II), Zn(II) and Ca(II)) is also determined by means of potentiometric studies. The prevailing species are [M(TirH)2]2+ where the ligand coordinates the metal ion through carboxylic group, while the piperidine nitrogen is still protonated. The great stability of these complexes may be due to the presence of hydrogen bond interactions, as well as the formation of stacking interactions involving the phenyl ring of the tyrosine residue.     

Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase

Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25 degrees C, and the dissociation constant for loss of CH2-H4folate from the noncovalent ternary complex is approximately 1 microM. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, delta G+ = 17.9 kcal/mol, delta H+ = 19.3 kcal/mol, and delta S+ = 0.005 kcal/(mol.deg). The equilibrium constant between the noncovalent and covalent ternary complexes is approximately 2 X 10(4), and the overall dissociation constant of CH2-H4folate from the covalent complex is approximately 10(-11) M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiazolium C(2)-proton exchange: structure-reactivity correlations and the pKa of thiamin C(2)-H revisited

Rate constants for C(2)-proton exchange from thiamin, N(1')-methylthiamin, and several 3-substituted-4-methylthiazolium ions catalyzed by D2O and deuterioxide ion were determined by 1H NMR at 30 degrees C and ionic strength 2.0 M. Values of pKa for the thiazolium ions, including thiamin itself, were found to be in the range pKa = 17-19; the pKa values for N(1')-protonated thiamin and free thiamin C(2)-H in H2O are 17.7 and 18.0, respectively. The pKa value for N(1')-protonated thiamin was calculated from the observed rate constant for the pD-independent reaction with D2O after correction for a secondary solvent deuterium isotope effect of kH2O/kD2O = 2.6. The pKa value for free thiamin was calculated from the rate constant for catalysis by OD- after correction by a factor of 3.3 = 8/2.4 for an 8-fold negative deviation of kOD from the Br?nsted plot of slope 1.0 for general base catalysis and a secondary solvent isotope effect of kOD/kOH = 2.4. Values of k-a = 2 X 10(10) and 3 X 10(9) M-1 s-1 were assumed for diffusion-controlled protonation of the C(2) ylide in the reverse direction by H3O+ and H2O, respectively. The Hammett rho I value for the exchange reaction catalyzed by deuterioxide ion or D2O is 8.4 +/- 0.2. There is no positive deviation of the rate constants for free or N(1')-substituted thiamin analogues in either Hammett correlation. This shows that the aminopyrimidinyl group does not provide significant intramolecular catalysis of nonenzymic C(2)-proton removal in the coenzyme.     

Investigation of the ternary D-myo-inositol 1,2,6-tris(phosphate)-spermine-Zn2+ system in solution

Interactions of Ins(1,2,6)P3 (IP), with spermine (Spm) and zinc cations have been studied by potentiometric and 31P NMR titrations. In the 4-11 pH range, two IPSpmZn2H3 and IPSpmZn2H mixed complexes are formed which are largely predominant with respect to the binary species. According to 31P NMR titration it is likely that one of the zinc cations preferably binds phosphates P1 and P6. The adduct formation between Ins(1,2,6)P3 and spermine seems also favourable to the formation of the mixed complexes. The occurrence of ternary complexes involving inositol-phosphates, biogenic amines, and metallic cations may be of relevance in the regulation of biological processes.     

(Model) studies on vanadate-dependent bromo/iodoperoxidase from Ascophyllum nodosum. VO2+ is not incorporated into the active site.

Vanadate-dependent peroxidase A.n.I, the main isoenzyme (M(r) = 100 kDa) from the seaweed, Ascophyllum nodosum, contains 2 V per enzyme molecule (as shown by ICP-MS metal analysis) after complete reconstitution with vanadate (V), possibly distributed in a 1:1 ratio between the surface and active site. VO2+ is only weakly associated to the surface of A.n.I. There is no transport channel for VO2+. The EPR spectrum of the reduced holoenzyme is anisotropic (axial) already at room temperature, with EPR parameters similar to those of VO2+ complexes of small model peptides such as Ala-His, Gly-Tyr, Gly-Ser, Gly-Glu, Ser-Gly and Phe-Glu. The complex formation between Ala-His and H2VO4- in water has also been investigated (by 51V NMR); the formation constant at pH 7.2 amounts to 266(28) M-1.     

Ethylenediamine-palladium(II) complexes with pyridine and its derivatives: synthesis, molecular structure and initial antitumor studies.

The synthesis of four mononuclear palladium complexes of general formula [Pd(en)Cl(L)]NO3 (en = ethylenediamine; L = pyridine (I), 4-methylpyridine (II), 4-hydroxypyridine (III) or 4-aminopyridine (IV) has been achieved. The structure of these compounds was studied by elemental analysis, IR, far-IR and 1H NMR; complex I was analyzed by X-ray diffraction. The crystal of [Pd(en)(pyridine)Cl]NO3 is monoclinic, space group P21/c (a = 7.990(2), b = 16.058(3), c = 9.846(2) A, beta = 103.81(3) degrees, Z = 4, R = 0.067, Rw = 0.066). The Pd(II) atom exhibits an approximately square planar coordination with bond lengths in the range 2.017-2.042 A for Pd-N and 2.320 A for Pd-Cl. In order to determine the donor strength of the aromatic pyridine ligands, the stability constants of binary complex ML2+ (M = [Pd(en) (H2O)2]2+; L = pyridine, 4-Me-pyridine, 4-OH-pyridine and 4-NH2-pyridine) were determined by potentiometric pH titration in aqueous solution (T = 25 degrees C, I = 0.1 mol l-1 NaNO3). The results show that the stability constants of the binary complexes systematically increase with increasing pKa of the pyridines. The above four palladium complexes, [Pt(en)(pyridine)Cl]NO3 and cis-diamminedichloroplatinum (II) (cis-DDP) were assayed for cytotoxicity in vitro against the human leukemia cell line HL-60, and compounds I, II, III and cis-DDP show significant cytotoxic activity against HL-60.     

Two γ-Methyl Biosides from Dregea volubilis (L.) Benth

From the hydrolysate of the crude glycosides from the roots, of Dregea volubilis(L.) Benth in Dehong, Yunnan, two α-methyl biosides Ⅰ and Ⅱ (yields: 0.016%and 0.0097%, respectively) were isolated by silica gel column chromatography. Theirchemical structures were established by interpretation of MS, IR,1H,13C-NMR, andgas chromatographic analysis of their degradation products, and comparison of thephysical properties of Ⅰ, Ⅱ and their acetates which were reported in literatures asfollows: α-methyl-pachybioside for Ⅰ, and α-methyl-[3-O-methyl-6-deoxy-D-allose(1→4)-D-olivoside] for Ⅱ. Ⅱ named α-methyl-dredehongbioside, is reported for the first time. Ⅱ, α-methyl-dredehongbioside, colorless needles (from MeOH), bitter, mp. 184–186℃,[α]D22 +74.5˚~(c= 0.52, MeOH). Anal. Cald(%) for C14H26O8:C52.17, H8.07;Found; C52.23,H8.22. Irvmaxkbr: 3370, 1443, 1419, 1375, 1268, 1218, 1168,1127,1060cm-1. MS(m/e,%): 322(M+,3),291(M+-OCH3,15), 273(M+-OCH3-H2O,12), 258,246,232, 222, 159, 145, 141, 128, 95, 87, 85, 74 (base peak, 100), 59. 1H NMRδ(CDCl3): 4.73(1H, dd, J= 4.0 Hz, J= 1.5Hz, C-1-H), 4.55(1H, d, J= 8.0Hz,C-1′-H), 3.79(1H, dd, J=3.0Hz, J= 3.0Hz, C-3′-H), 3.00(1H, dd, J= 9.0Hz,J= 9.0Hz, C-4-H), 2.22(1H, m, C-2-Ha), 1.60(1H, m, C-2-He), 1.33(3H, d,J= 6.0Hz,C-5-CH,), 1.31(3H,d,,J= 6.5Hz, C-5′-CH), 3.68(3H,s,,C-3′-OCH),3.31(3H,s,C-1-OCH). 13C NMR data were seen in Table 1. Ⅳ, tri-acetyl-α-methyl-dredehongbioside, colorless granular (from MeOH),mp. 135--137℃, [a]D22+ 88.2˚(c= 0.50, MeOH). MS(m/e, %): 488 (M+, 2), 388(M+-HOAc,2), 357(M+-OCH3-HOAc,33), 288, 187, 127, 116, 85, 74, 59, 43(basepeak, 100). 1H NMR,δ(CDCl3): 5.25(1H, ddd,.J= 11.0Hz, J=9.0Hz,.J= 5.5Hz,C-3-H), 4.86(1H, d, J= 8.0Hz, C-1′-H), 4.69 (1H, dd, J= 4.0Hz, J= 1.5Hz,C-1-H), 4.58(1H,m,C-2′-H),3.94(1H, dd, J= 3.0Hz,J= 3.0Hz,C-3′-H),3.64(1H,m,C-5-H), 3.22(1H,dd, J= 9.5Hz, J= 8.5Hz, C-4-H), 2.30(1H, m, C-2-Ha),1.67(1H,m,C-2-He), 1.31(3H, d, .J= 6.5Hz, C-5-CH3), 1.17(3H, d, J= 6.0Hz,C-5-CH3), 3.47(3H, s, C-3′-OCH3), 3.30(3H,s, C-1-OCH3), 2.10(6H, s, C-2′, C- 4′ -OCH3), 2.03(3H,s,C-3-OCH3).     

Aquo-pentamine Co(III) complexes as models for carbonic anhydrase

The hydrolysis of 4-nitrophenyl acetate by metal complexes Co(en)2(imH)H2O3+, Co(en)2(bzmH)H2O3+, and Co(en)2(imCH3)H2O3+ (imH = imidazole, bzmH = benzimodazole, imCH3 = methyl imidazole) has been investigated in the pH range 5.4-8.9. The small difference in nucleophilic reactivity in the pH range 5.4-6.7 is assumed to be due to hydrogen bonding abilities of the imidazole and substituted imidazole ligands and small pKa differences (k2(imH) = 2.2 X 10(-2) M-1 sec-1, k2(bzmH) = 5.68 X 10(-2) M-1 sec-1, k2(imCH3) = 1.35 X 10(-2) M-1 sec-1, 40 degrees C, 1 = 0.3 NaClO4, pKa(imH) = 6.2, pKa(imCH3) = 6.2 and pKa(bzmH) = 5.9). In the pH range 7.8-8.9, the differences in nucleophilic reactivity (k3(imH) = 85.5 X 10(-2) M-1 sec-1, k3(bzmH) = 33.4 X 10(-2) M-1 sec-1, 40 degrees C, I = 0.3 NaClO4) are reconciled with a significant steric factor outweighing the acidity of the benzimidazole complex. In the pH region 6.7-7.7, the deviation from linearity is presumably due to both hydroxo and imido ligands functioning as nucleophiles, the latter being about 40 times stronger than the former.     

Ligand-exchangeability of 2-coordinate phosphinegold(I) complexes with AuSP and AuNP cores showing selective antimicrobial activities against Gram-positive bacteria. Crystal structures of [Au(2-Hmpa)(PPh(3))] and [Au(6-Hmna)(PPh(3))] (2-H(2)mpa=2-mercaptopropionic acid, 6-H(2)mna=6-mercaptonicotinic acid)

Selective and effective antimicrobial activities against Gram-positive bacteria (B. subtilis and/or S. aureus) were found in 2-coordinate gold(I)-PPh(3) complexes with AuSP and AuNP cores, i.e. [Au(L)(PPh(3))] (HL=2-H(2)mna [H(2)mna=mercaptonicotinic acid] 3, D-H(2)pen [H(2)pen=penicillamine] 4, D,L-H(2)pen 5, 4-H(2)mba [H(2)mba=mercaptobenzoic acid] 8, Hpz [Hpz=pyrazole] 9, Him [Him=imidazole] 10, 1,2,3-Htriz [Htriz=triazole] 11, 1,2,4-Htriz 12, Htetz [Htetz=tetrazole] 13), whereas no activity was observed in 2-coordinate AuSP core complexes [Au(2-Hmba)(PPh(3))] 6 and [Au(3-Hmba)(PPh(3))] 7. The two novel AuSP core complexes, [Au(2-Hmpa)(PPh(3))] [H(2)mpa=mercaptopropionic acid] 1 and [Au(6-Hmna)(PPh(3))] 2, were prepared and characterized by elemental analysis, FT-IR, TG/DTA, and ((31)P, 1H and 13C) NMR spectroscopy. The crystal structures of 1 and 2 were determined as a supramolecular arrangement of the 2-coordinate AuSP core. Both 1 and 2 significantly showed antibacterial activities. As a model reaction of phosphinegold (I) complexes with the cysteine residue in the biological ligands, we examined if the ligand exchange reactions of the aromatic anions L(1)(-) in [Au(L(1))(PPh(3))] (HL(1)=6-H(2)mna 2, 2-H(2)mna 3, 2-H(2)mba 6, Hpz 9, Him 10, 1,2,3-Htriz 11, 1,2,4-Htriz 12) with aliphatic thiols HL(2) (HL(2)=2-H(2)mpa, D-H(2)pen) occurred under the mild conditions and, also, if the 'reverse' reactions, namely, the ligand exchange reactions of the thiolate anions in [Au(2-Hmpa)(PPh(3))] 1, [Au(D-Hpen)(PPh(3))] 4 and [Au(2-Hmba)(PPh(3))] 6 with the free ligands HL(1) took place under similar conditions. In this work, a relationship of the ligand-exchangeability among 2-coordinate gold(I) complexes (1-4, 6, 9-12) was revealed. Complex 6 was substitution-inert, whereas complexes 1-4 and 9-12 were substitution-labile. The ligand-exchangeability of Au-S and Au-N bonds in the 2-coordinate phosphinegold(I) complexes with AuSP and AuNP cores to form new AuSP cores, with retention of the Au-P bond, was closely related to the observed activities against Gram-positive bacteria, and the ease of the ligand-exchange reaction was strongly related to the intensity of the activities.     

Antibiotic as ligand. Coordinating behavior of the cephalexin towards Zn(II) and Cd(II) ions

The complex formation equilibria of Zn(II) and Cd(II) with cephalexin have been studied through potentiometric titrations. Experimental data were analyzed using the least squares computer program SUPERQUAD. The stability constants were 1g beta ZnCEX+ = 2.40, 1g beta Zn(CEX)(OH) = -4.54, 1g beta CdCEX+ = 2.18, and 1g beta Cd(CEX)(OH) = -5.18 (I = 0.1 M NaNO3), CEX complexes of formulae Zn(CEX)2(3)H2O and Cd(CEX)(OH)H2O have been synthesized and characterized by elemental analysis, IR spectra, conductivity measurements, and electronic and NMR spectra. The thermal behavior of the synthesized compounds were studied by TGA and DTA. We conclude that the metal ion interacts with the amido group of CEX.     

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.     

Complexes of aluminium(III) with glucose-6-phosphate in aqueous solutions

The interaction of aluminium(III) with glucose-6-phosphate (GP: LH2) in aqueous solutions has been studied from pH 1 to pH 8, by pH-potentiometry and multinuclear (31P, 27Al, 13C) NMR spectroscopy. Various mononuclear species (MLH2, MLH, ML, ML2H, ML2 and MLH(-3)) and dinuclear complexes M2L2H-n (n=1-4) are formed in the system. NMR clearly indicates that GP is already bound to Al(III) at pH 1. The potentiometric speciation results are confirmed and completed by spectroscopic experiments. Many peaks are observed in the 31P NMR spectra suggesting the formation of isomeric species. An attempt to assign the signals to the corresponding complexes is made, allowing a discussion about their structure. Interestingly enough no metal ion-induced deprotonation and coordination of the alcoholic-OH functions have been observed.     

Spectrophotometric and ESR evidence for vanadium(IV) deferoxamine complexes.

Complexes of vanadium(IV), vanadyl, are reported to be formed with the trihydroxamic acid deferoxamine (H3DF+). One complex exhibits a reddish-violet color, with a major absorbance peak at 386 nm and a smaller peak at 520 nm. This complex is potentially useful for the microdetermination of vanadyl. The apparent molar absorptivity is 3.91 mM-1 cm-1, and the complex obeys Beer's law in the concentration range of 0.6-63 ppm. Electron spin resonance studies indicate the formation of two vanadyl complexes that are 1:1 in vanadyl and deferoxamine, but have two or three bound hydroxamate groups. ESR and spectrophotometric evidence indicate that the red, low pH form, involves an octahedral vanadium (4+) ion coordinated by three hydroxamate ligands. One of these hydroxamates is displaced by an oxygen at pH greater than 2.8 according to the following equilibria: VO2+ + H3DF+ in equilibrium with VIV(DF)2+ + H3O+, VIV(DF)2+ + H2O in equilibrium with VO(HDF)+ + H+, where pk2 = 2.8.     

Ligational behavior of two biologically active N-S donors toward oxovanadium(IV) ion and potentiation of their antibacterial activities by chelation to metal ions. Part III

Chelating behavior of two biologically active ligands, pyridine-2-carboxaldehyde thiosemicarbazone (PT) and pyridine-2-carboxaldehyde-(4-phenyl)thiosemicarbazone (PPT), toward oxovanadium(IV) ion has been studied. The ligands are found to react in the thioketo form (pH 2-4), yielding the complexes [VO(PT)X2](X = Cl-, Br-, ClO4-), [VO(PT)(SO4)H2O], [VO(PPT)2X]X (X = Cl-, Br-, ClO4-) and [VO(PPT)2SO4]. Reactions of [VO(PT)(SO4)H2O] and [VO(PPT)2X]X (X = Cl-, Br-, ClO4-) with a monodenate Lewis base (B) like pyridine lead to the formation of [VO(PT)(SO4)Py]H2O and [VO(PPT)2py]X2 respectively. Bonding sites of the donor molecules around the oxometal cation have been located. Nature of the EPR spectra and magnetic moment values point to the monomeric character of the complexes and suggest a distorted octahedral donor environment for the oxovanadium(IV) ion. Status of the metal-oxygen multiple bond in all the complexes has been computed in terms of the V-O(1) stretching force constant. The ligands themselves and most of their oxovanadium(IV) complexes are found to exert powerful in vitro antibacterial activities towards E. coli.     

Hg(II)-coordination by sugar-acids: role of the hydroxy groups

A solution study on the ability of some derivatised sugars [glucuronic acid (GluA), galacturonic acid (GalA) and glucosaminic acid (GlNA)] to complex the Hg(II) ion is reported. The stability constants of the complex species were determined by potentiometric measurements while (1)H NMR experiments allow to define the coordination sites of sugar molecules. GluA coordinates the metal ion through the carboxylic oxygen and the O-4 hydroxyl group and is found to form more stable complexes with respect to GalA in which metal ligation is from the carboxylic oxygen and the O-5 ring oxygen. GlNA forms stable complexes chelating Hg(II) ion through carboxylic oxygen and the alpha-amino group. The ternary 2,2'-bipyridine containing systems were also investigated by means of potentiometric studies. The ML(2) complexes were also isolated in the solid state and characterised by IR spectroscopy.     

Hydrogen-1 nuclear magnetic resonance studies of staphylococcal nuclease variant H124L: pH dependence of histidines and tyrosines

The pH dependence of the 1H NMR spectrum of staphylococcal nuclease H124L was investigated as a function of the binding of Ca2+, the ion required for enzymatic activity, and deoxythymidine-3',5'-diphosphate (pdTp), a competitive inhibitor. The protein studied was the product of a cloned gene expressed in Escherichia coli which yields a protein having a sequence identical to that of the nuclease isolated from the V8 strain of Staphylococcus aureus. Of the observable ring protons of the three histidine residues, only the C delta 1H of His46 shows a large chemical shift perturbation on formation of the ternary complex, (nuclease H124L).pdTp.Ca2+. The pKa of His46 is lowered by 0.2 pH unit in the binary complex. All seven tyrosines titrate with normal pKa values between 9 and 11 in the unligated nuclease. In the ternary complex, however, the pKa values of Tyr85 and Tyr93 increase above pH 11.0. The chemical shift perturbations of the ring protons of the Tyr27, Tyr85, Tyr113, and Tyr115 were observed between pH 4 and 6; these spectral perturbations are attributed to interactions with carboxylate groups. Binding Ca2+ alone acted opposite to the perturbation in Tyr113 and Tyr115. Ca2+ binding leads to deshielding the ring protons of Tyr113, but this effect is removed in the ternary complex. Binding of pdTp and Ca2+ stabilizes the protein against high pH denaturation up to pH 11.5.     

Kinetic analysis of the acid-alkaline conversion of horseradish peroxidases.

The nature of the acid-alkaline conversion of horseradish peroxidases was studied by measuring four rate constants in reactions, E + H+ (k1) in equilibrium (k2) EH+ and E + H2O (k3) in equilibrium (k4) EH+ + OH-, where EH+ and E denote the acid and alkaline forms of the enzymes. The values of k1, (k2 + k3), and k4 were obtained by measuring the relaxation rates of the acid leads to alkaline and alkaline leads to acid conversions by means of th pH jump method with a stopped-flow apparatus. The value of k3 could also be obtained by measuring the rate of reactions between hydrogen peroxide and peroxidases at alkaline pH. The measurements were conducted with four peroxidases having different pKa values: peroxidase A )pKa = 9.3), peroxidase C (pKa = 11.1), diacetyldeuteroperoxidase A (pKa = 7.7), and diacetyldeuteroperoxidase C (pKa = 9.1). The value of k1 was about 10(10) M-1 s-1 in the reaction of the four enzymes while k4 was quite different between the enzymes. The pKa was determined by k3 and k4 for the natural peroxidases and by k1 and k2 for the diacetyldeuteroperoxidases. The mechanism of the acid-alkaline conversion was discussed in comparison with that of metmyoglobin.     

The role of protons in the mechanism of galactoside transport via the lactose permease of Escherichia coli

The kinetic mechanism of lactose transport across the cytoplasmic membrane has been investigated and the results related to standard models for the lactose-H+ symport reaction using computer simulation. It is shown that the biphasic kinetics reported for lactose uptake (Kaczorowski, G.J. and Kaback, H.R. (1979) Biochemistry 18, 3691-3697) are consistent with random binding of lactose and protons and rapid subsequent translocation of the ternary lactose-H+-permease complex. Such a model is also shown to explain the observed dependence of the kinetic parameters on the magnitude of the protonmotive force. Both sugar and protons are shown to cause product inhibition of lactose flux and the ability of standard models to account for the pattern of inhibition is discussed. Three apparent dissociation constants have been determined for the protonation reactions in the external medium: two (pKa 6.3 and 9.6) control the activity of the permease, whilst the third (pKa 8.3) controls the affinity of the permease for galactosides. A similar set of dissociation constants has been determined for the internal reactions. Again two (pKa 6 and 9.8) control activity and a third (pKa 8.8) controls the affinity for galactosides. The dissociation reactions characterised by pKa 8.3, 8.8, 9.6 and 9.8 are attributed to the dissociation of the substrate (symported) proton from the binary proton-permease complexes (pKa 8.3 and 8.8) and the ternary proton-galactoside-permease complexes (pKa 9.6 and 9.8). The third pair (pKa 6.3 and 6.0) must be interpreted as describing a separate protonation reaction which may have a regulatory or auxiliary role in transport.     

Synthesis and reactivity of the oxovanadium(IV) complexes of two N-O donors and potentiation of the antituberculosis activity of one of them on chelation to metal ions: Part IV

A new series of oxovanadium(IV) complexes of two aromatic acidhydrazides (BH and AH) have been reported. Of these two donors, AH is known to possess considerable in vitro antitubercular activity. At pH 2-4, oxometal complexes of the type [VO(BH/AH)2SO4].nH2O (n = 1, 0) and [VO(BH/AH)(C2O4)H2O].H2O (BH = C6H5CONHNH2 and AH = (2-NH2)C6H4.CO.NHNH2) were obtained. Reactions of [VO(BH/AH)(C2O4)H2O].H2O with a monodentate Lewis base lead to the isolation of metal-ligand complexes [VO(BH/AH)(C2O4)L].nH2O (L = NH3, n = 1, L = py, n = 2). Disposition of the bonding sites of donor molecules around the oxometal acceptor center and status of the metal-oxygen multiple bond have been established. A monomeric and distorted octahedral donor environment for the oxovanadium(IV) ion has been proposed on the basis of the electron paramagnetic resonance (EPR) spectra and magnetic susceptibility measurements. Antitubercular activities, in vitro, of the oxovanadium(IV) complexes of AH have also been evaluated towards tuberculosis mycobacteria such as Mycobacterium flae, Mycobacterium smegmatis and Mycobacterium H37Rv.     

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.