Manganese, calcium, and saccharide binding to concanavalin A, as studied by ultrafiltration

The binding of the ligands Mn2+, Ca2+, and methyl alpha-D-glucopyranoside to concanavalin A, purified as described (A.J. Sophianopoulos and J.A. Sophianopoulos (1981) Prep. Biochem. 11, 413-435), was studied by ultrafiltration in 0.2 M NaCl, pH 5.2 and pH 6.5 to 7, and at 23 to 25 degrees C. The association constant (Ka) of methyl alpha-D-glucopyranoside to concanavalin A was (2 +/- 0.2) X 10(3) M-1, both at pH 5.2 and 7. At pH 5.2 and in the absence of Ca2+, the Ka of Mn2+ to concanavalin A was (5 +/- 1) X 10(3) M-1, and in the presence of 1 mM Ca2+, the Ka was (9.1 +/- 2.1) X 10(5) M-1. At pH 6.5 Mn2+ bound to concanavalin A with a Ka of (7.3 +/- 1.8) X 10(5) M-1, and the binding affinity was virtually independent of the presence of Ca2+. Experiments of binding of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A indicated that at pH 5.2, binding of a single Mn2+ per concanavalin A monomer was sufficient to induce a fully active saccharide binding site. Ca2+ is not necessary for such activation, but rather it increases the affinity of concanavalin A for binding Mn2+.     

Electron spin echo envelope modulation studies of lectins: evidence for a conserved Mn(2+)-binding site.

Electron spin echo envelope modulation (ESEEM) experiments have been used to investigate the Mn(2+)-binding site in a series of lectins including concanavalin A, pea lectin (Pisum sativum), isolectin A from lentil (Lens culinaris), soybean agglutinin (Glycine max), Erythrina indica lectin, and Lotus tetragonolobus isoelectin A. Together with model studies, the results provide direct evidence for a single nitrogen atom of a conserved residue bonded directly to Mn2+ in all of them. ESEEM measurements of the lectins exchanged with deuterium oxide, together with model studies, provide evidence for the presence of two water molecules coordinated to the Mn2+ in all of the proteins. In contrast to concanavalin A, the absence of solvent exchange at the Mn2+ site in the pea and lentil lectins demonstrated by nuclear magnetic relaxation dispersion measurements [Bhattacharyya, L., Brewer, C.F., Brown, R. D., III, & Koenig, S. H. (1985) Biochemistry 24, 4985-4990] must therefore be due to slow exchange of the water ligands of the bound Mn2+. Binding of saccharides was observed to have little effect on the structural features of the Mn2+ site in the lectins as determined by ESEEM.     

Proton and deuteron nuclear magnetic relaxation dispersion studies of Ca2+-Mn2+-lentil lectin and Ca2+-Mn2+-pea lectin: evidence for a site of solvent exchange in common with concanavalin A

Measurements of the magnetic field dependence of the longitudinal magnetic relaxation rates (NMRD profiles) of solvent protons and deuterons led to the discovery of two classes of solvent binding sites in Ca2+-Mn2+-concanavalin A (CMPL) [Koenig, S. H., Brown, R. D., III, & Brewer, C. F. (1985) Biochemistry (second of three papers in this issue)]. In this paper, we compare proton and deuteron NMRD profiles of Ca2+-Mn2+-lentil lectin (CMLcH) and Ca2+-Mn2+-pea lectin (CMPSA) with those of CMPL. All three metalloproteins are D-mannose/D-glucose-specific lectins that have a high degree of structural similarity and require the metal ions for their biological activities. We have developed a method for the preparation of fully active metal ion derivatives of lentil lectin (LcH) and pea lectin (PSA), including the diamagnetic derivatives Ca2+-Zn2+-LcH and Ca2+-Zn2+-PSA [Bhattacharyya, L., Brewer, C. F., Brown, R. D., III, & Koenig, S. H.(1984) Biochem. Biophys. Res. Commun. 124, 857-862]. The behavior of these two lectins with regard to their NMRD profiles is essentially identical, for both the paramagnetic and diamagnetic forms. Together with CMPL, all three lectins have a common paramagnetic contribution with a negative temperature dependence of the rates, while CMPL contributes an additional component with a positive temperature dependence. The common contribution derives from the class of fast exchanging water molecules observed in the proton NMRD profile of CMPL (Koenig et al., 1985); their protons are calculated to be relatively remote from the Mn2+ ions (4.4 A for CMPL and 5.5 A for LcH and PSA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calculation of free-Mg2+ concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo

We have attempted to resolve the differences between the levels of free Mg2+ in muscle calculated by Wu et al. [Wu, S. T., Pieper, G. M., Salhany, J. M., & Eliot, R. S. (1981) Biochemistry 20, 7399-7403] (2.5 mM in guinea pig heart) and by Gupta and Moore [Gupta, R. K., & Moore, R. D. (1980) J. Biol. Chem. 255, 3987-3993] (0.6 mM in frog skeletal muscle) on the basis of substantially identical measurements by 31P NMR of the phosphate peaks in the spectrum of MgATP2-. The differences depend on the methods of calculation, including which reactions in which multiple equilibria are being considered. Biochemists and physical chemists customarily use different working definitions of the stability constant for MgATP2- in particular. Wu et al. used in their calculations, without reconciliation, methods involving three different operational definitions of the chelation equilibria involved. An algorithm for calculating Mg2+ and total ATP, which can be carried out with a hand calculator, is described here. With it, we calculated Mg2+ levels that agree with those determined by Gupta et al. [Gupta, R. K., Benkovic, J. L., & Rose, Z. B. (1978) J. Biol. Chem. 253, 6165-6171] with their in vitro systems. We therefore agree with the finding of Gupta and Moore that the Mg2+ level in skeletal and cardiac muscle is 0.6 mM.     

Negative cooperativity associated with binding of multivalent carbohydrates to lectins. Thermodynamic analysis of the "multivalency effect".

Our previous study demonstrated that isothermal titration microcalorimetry (ITC) could be used to determine the thermodynamics of binding of a series of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) [Dam, T. K., Roy, R., Das, S. K., Oscarson, S. and Brewer, C. F. (2000) J. Biol. Chem. 275, 14223-14230]. The higher affinities of the multivalent carbohydrates for the two lectins were shown to be due to their greater positive entropy of binding contributions relative to monovalent analogues. In the present study, ITC data from our previous report for binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues to ConA and DGL were subjected to Hill plot analysis. Hill plots of the binding of monovalent methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside to ConA and DGL are linear with slopes near 1.0, demonstrating a lack of binding cooperativity and allosteric transitions in the proteins. However, Hill plots for the binding of the di-, tri-, and tetravalent trimannoside analogues to both lectins are curvilinear with decreasing tangent slopes below 1.0, indicating increasing negative cooperativity upon binding of the analogues to the lectins. The curvilinear Hill plots are consistent with decreasing affinity and functional valencies of the multivalent analogues upon sequential binding of lectin molecules to the carbohydrate epitopes of the analogues. The following paper [Dam, T. K., Roy, R., Pagé, D., and Brewer, C. F. (2002) Biochemistry 41, 1359-1363] provides direct evidence of the decreasing affinity constants of multivalent carbohydrates upon sequential binding of lectin molecules.     

Stoichiometry of manganese and calcium ion binding to concanavalin A

Using measurements of solvent nuclear (proton) magnetic relaxation dispersion (NMRD), we have previously shown that concanavalin A (Con A) can exist in two conformational forms and that, in the absence of Ca2+, Mn2+ can bind to both the S1 and S2 sites of each monomer of Con A of at least one conformer [Brown, R.D., III, Brewer, C.F., & Koenig, S.H. (1977) Biochemistry 16, 3883-3896]. Recently other investigators have claimed that the stoichiometry of Mn2+ binding to Con A is only 1:1 for this conformational state, both in the absence and presence of saccharide; the same was claimed for Ca2+ under similar conditions. We now present titration and equilibrium dialysis experiments, both in the absence and presence of saccharide, using NMRD and atomic absorption spectroscopy, to investigate the stoichiometry of Mn2+ and Ca2+ binding to Con A. We have extended the NMRD method to include the determination of the total concentration of Mn2+ in samples of Con A. This, coupled with our previous use of NMRD to measure the concentration of free Mn2+ in protein solutions as well as the distribution of bound Mn2+ among different sites, allows us to measure the stoichiometry of binding with precision. We reconfirm that, at equilibrium in the presence of excess Mn2+, the binding stoichiometry of Mn2+ to Con A is 2:1, both in the absence and presence of saccharide. Addition of Ca2+ to a solution of Mn2+-Con A results in stoichiometric displacement of Mn2+ from the S2 site under the conditions investigated. Under nonequilibrium conditions, Mn2+ forms a metastable binary complex with the protein that persists for days at 5 degrees C. We also report, for the first time, values for all of the dissociation constants of binary and ternary complexes of Mn2+ with both conformations of Con A in solution. Atomic absorption measurements also indicate that Ca2+, in the absence of Mn2+, binds to both S1 and S2 sites in the absence and presence of saccharides.     

Proton release due to manganese binding and oxidation in modified bacterial reaction centers

The pH dependence of binding and oxidation of Mn2+ in highly oxidizing reaction centers with designed metal-binding sites was characterized by light-minus-dark optical difference spectroscopy and direct measurements of proton uptake/release. These mutants bind a Mn2+ ion that can efficiently transfer an electron to the oxidized bacteriochlorophyll dimer, as described earlier [Thielges et al. (2005) Biochemistry 44, 7389-7394]. The dissociation constant, KD, significantly increased with decreasing pH. The pH dependence of KD between pH 7 and pH 8 was consistent with the binding of Mn2+ being stabilized by the electrostatic release of two protons. The strong pH dependence of proton release upon Mn2+ binding, with a maximal release of 1.4 H+ per reaction center, was interpreted as being a result of a shift in the pKa values of the coordinating residues and possibly other nearby residues. A small amount of proton release associated with Mn2+ oxidation was observed upon illumination. These results show that functional metal-binding sites can be incorporated into proteins upon consideration of both the metal coordination and protonation states of the ligands.     

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.     

Thermodynamic binding parameters of individual epitopes of multivalent carbohydrates to concanavalin a as determined by "reverse" isothermal titration microcalorimetry.

The preceding paper [Dam, T. K., Roy, R., Pagé, D., and Brewer, C. F. (2002) Biochemistry 41, 1351-1358] demonstrated that Hill plots of isothermal titration microcalorimetry (ITC) data for the binding of di-, tri-, and tetravalent carbohydrate analogues possessing terminal 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside residues to the lectin concanavalin A (ConA) show increasing negative cooperativity upon binding of the analogues to the lectin. The present study demonstrates "reverse" ITC experiments in which the lectin is titrated into solutions of di- and trivalent analogues. The results provide direct determinations of the thermodynamics of binding of ConA to the individual epitopes of the two multivalent analogues. The n values (number of binding sites per carbohydrate molecule) derived from reverse ITC demonstrate two functional binding epitopes on both the di- and trivalent analogues, confirming previous "normal" ITC results with the two carbohydrates [Dam, T. K., Roy, R., Das, S. K., Oscarson, S., and Brewer, C. F. (2000) J. Biol. Chem. 275, 14223-14230]. The reverse ITC measurements show an 18-fold greater microscopic affinity constant of ConA for the first epitope of the divalent analogue versus its second epitope and a 53-fold greater microscopic affinity constant of ConA binding to the first epitope of the trivalent analogue versus its second epitope. The data also demonstrate that the microscopic enthalpies of binding of the two epitopes on the di- and trivalent analogues are essentially the same and that differences in the microscopic K(a) values of the epitopes are due to their different microscopic entropies of binding values. These findings are consistent with the increasing negative Hill coefficients of these analogues binding to ConA in the previous paper.     

Formation of highly ordered cross-linked lattices between asparagine-linked oligosaccharides and lectins observed by electron microscopy

The interaction of asparagine-linked carbohydrates (N-linked) with carbohydrate binding proteins called lectins has been demonstrated to be involved in a variety of cellular recognition processes. Certain N-linked carbohydrates have been shown to be multivalent and capable of binding, cross-linking, and precipitating lectins (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1988) Biochemistry 27, 1034-1041). Recent data have further suggested that certain oligomannose and bisected hybrid-type N-linked glycopeptides form homogeneous cross-linked lattices with concanavalin A (Bhattacharyya, L., Khan, M. I., and Brewer, C. F. (1988) Biochemistry 27, 8762-8767). In the present study, evidence has been obtained from electron microscopy for the formation of highly ordered and distinct lattices for two bivalent complex type oligosaccharides cross-linked with soybean lectin (Glycine max) and isolectin A from Lotus tetragonolobus, respectively. The results indicate a new source of specificity for interactions of N-linked carbohydrates with lectins, namely their ability to form highly ordered homogeneous aggregates.     

Biosynthesis of P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate in calf pancreas microsomes

Calf pancreas microsomes incubated with UDP-N-acetyl-D-[14C] glucosamine in the presence of Mn2+ incorporated radioactivity into P1-2-acetamido-2-deoxy-D-glucopyranosyl P2-dolichyl pyrophosphate and P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate. The formation of both glycolipids was enhanced to the same extent by exogenous dolichyl phosphate. Labeled P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate was formed from synthetic P1-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P2-dolichyl pyrophosphate and from prelabeled pancreatic P1-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P2-dolichyl pyrophosphate without the addition of divalent cation. Upon thin layer chromatography, it had the same mobility as synthetic P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate recently synthesized by Warren et al. (Warren, C. D., Herscovics, A., and Jeanloz, R. W. (1977) Carbohydr. Res., in press), but was different from the synthetic compound prepared by Wedgwood et al. (Wedgwood, J. F., Warren, C. D., Jeanloz, R. W., and Strominger, J. L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 5022-5026).     

Fluorescence study of brevin, the Mr 92 000 actin-capping and -fragmenting protein isolated from serum. Effect of Ca2+ on protein conformation

The fluorescence characteristics of brevin and the effects of Ca2+ on the protein conformation were fully investigated. Brevin contains 18 tryptophans and 27 tyrosines. Analysis of the fluorescence spectra and the accessibility to quenching molecules indicate that the emitting tryptophans are located in a hydrophobic environment (lambda max = 324 nm) close to the protein surface. In native brevin, tyrosyl residues do not contribute to the fluorescence emission. Partial quenching of these chromophores has to be attributed to tyrosine----tryptophan resonance energy transfer which is highly efficient. The effect of brevin on actin polymerization has been shown to be Ca2+ sensitive [Harris, D. A., & Schwartz, J. H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6798-6802; Thorstensson, R., Utter, G., & Norberg, R. (1982) Eur. J. Biochem. 126, 11-16; Wilkins, J. A., Schwartz, J. H. & Harris, D. A. (1983) Cell Biol. Int. Rep. 7, 1097-1104; Harris, H. E., & Weeds, A. G. (1983) Biochemistry 22, 2728-2741] and brevin binding to hydrophobic matrices to be Ca2+ dependent (Z. Soua, personal communication). Ca2+ binding to brevin decreases the tryptophan fluorescence polarization degree (without affecting the excited-state lifetime), which suggests a higher chromophore mobility. This effect may be partly related to the slight unshielding of the tryptophan residues observed in fluorescence quenching experiments. Moreover, the reactivity of brevin sulfhydryl groups toward 5,5'-dithiobis(2-nitrobenzoic acid) increases in the presence of Ca2+. On the other hand, fluorescence spectra, quantum yields, excited-state lifetimes, and thermostability remain unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the concanavalin A-methyl alpha-D-mannopyranoside complex at 6-A resolution.

The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A-methyl alpha-D-mannopyranoside complex and is 35 A from the iodophenol binding site (K. D. Hardman and C. F. Ainsworth (1973), Biochemistry 12,4442), which has been postulated to be adjacent to the carbohydrate-specific binding site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 2580). The crystals are orthorhombic in space group C222(1) and crystal denisty measurements indicate a protein mass of four monomers (molecular weight of 104 000) per asymmetric unit. However, the electron density map contains eight monomers/asymmetric unit, revealing lattice disorder. The electron density map with a nominal resolution of 6 A has been solved using three heavy-atom derivatives and the position and orientation of each monomer established. Atomic coordinates of the native protein which has previously been determined (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103) were transposed into this new space group and the gross conformations of the monomers, dimers, and tetramers were found to be very similar to the previous structure. However, some minor differences were apparent even at this resolution. After crystal growth, the methyl alpha-D-mannopyranoside was replaced by o-iodophenyl beta-D-glucopyranoside or methyl 2-iodoacetimido-2-deoxy-alpha-D-glucopyranoside in separate experiments, and difference electron density maps were calculated. The highest peaks for both iodinated sugar derivatives associated with each monomer agreed within a few angstroms of each other and were found near side chains Tyr-12 and -100 and Asp-16 and -208. This region is 10-14 A from the manganese, in good agreement with nuclear magnetic resonance (NMR) studies in solution (C. F. Brewer et al. (1973), Biochemistry 12, 4448) and with the site predicted from crosslinked 1222 crystal studies (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103).     

Optimal salt bridge for Trp-cage stabilization

Gai and co-workers [Bunagan, M. R., et al. (2006) J. Phys. Chem. B 110, 3759-3763] reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 [Hudaky, P., et al. (2008) Biochemistry 47, 1007-1016]; the authors suggested that [D9E]-TC5b presented a more compact and melting resistant structure because of the "optimal distance between the two sides of the molecule". Nonetheless, the authors reported essentially the same circular dichroism (CD) melting temperature, 38 ± 0.3 °C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by nuclear magnetic resonance and CD with the following mutations: [D9E], [D9R,R16E], [R16O], [D9E,R16O], [R16K], and [D9E,R16K]. Of these, the [D9E] mutant displayed the smallest acidification-induced change in the apparent T(m). In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9-R16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence of salt bridge formation is also provided for a swapped pair, the [D9R,R16E] mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence that was cited as supporting increased fold stability for [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded states rather than a more favorable salt bridge. Our study also provides evidence of other Trp-cage stabilizing roles of the R16 side chain.     

pH optimum of the photosystem II H₂O oxidation reaction: effects of PsbO, the manganese-stabilizing protein, Cl- retention, and deprotonation of a component required for O₂ evolution activity

Hydroxide ion inhibits Photosystem II (PSII) activity by extracting Cl(-) from its binding site in the O(2)-evolving complex (OEC) under continuous illumination [Critchley, C., et al. (1982) Biochim. Biophys. Acta 682, 436]. The experiments reported here examine whether two subunits of PsbO, the manganese-stabilizing protein, bound to eukaryotic PSII play a role in protecting the OEC against OH(-) inhibition. The data show that the PSII binding properties of PsbO affect the pH optimum for O(2) evolution activity as well as the Cl(-) affinity of the OEC that decreases with an increasing pH. These results suggest that PsbO functions as a barrier against inhibition of the OEC by OH(-). Through facilitation of efficient retention of Cl(-) in PSII [Popelkova, H., et al. (2008) Biochemistry 47, 12593], PsbO influences the ability of Cl(-) to resist OH(-)-induced release from its site in the OEC. Preventing inhibition by OH(-) allows for normal (short) lifetimes of the S(2) and S(3) states in darkness [Roose, J. L., et al. (2011) Biochemistry 50, 5988] and for maximal steady-state activity by PSII. The data presented here indicate that activation of H(2)O oxidation occurs with a pK(a) of ～6.5, which could be a function of deprotonation of one or more amino acid residues that reside near the OEC active site on the D1 and CP43 intrinsic subunits of the PSII reaction center.     

Mechanism of inactivation of gamma-aminobutyrate aminotransferase by 4-amino-5-fluoropentanoic acid. First example of an enamine mechanism for a gamma-amino acid with a partition ratio of 0

The mechanism of inactivation of pig brain gamma-aminobutyric acid aminotransferase (GABA-T) by (S)-4-amino-5-fluoropentanoic acid (1, R = CH2CH2COOH, X = F) previously proposed [Silverman, R. B., & Levy, M. A. (1981) Biochemistry 20, 1197-1203] is revised. apo-GABA-T is reconstituted with [4-3H]pyridoxal 5'-phosphate and inactivated with 1 (R = CH2CH2COOH, X = F). Treatment of inactivated enzyme with base followed by acid denaturation leads to the complete release of radioactivity as 6-[2-hydroxy-3-methyl-6-(phosphonoxymethyl)-4-pyridinyl]-4-oxo-5-+ ++hexenoic acid (4, R = CH2CH2COOH). Alkaline phosphatase treatment of this compound produces dephosphorylated 4 (R = CH2CH2COOH). These results support a mechanism that was suggested by Metzler and co-workers [Likos, J. J., Ueno, H., Feldhaus, R. W., & Metzler, D. E. (1982) Biochemistry 21, 4377-4386] for the inactivation of glutamate decarboxylase by serine O-sulfate (Scheme I, pathway b, R = COOH, X = OSO3-).     

Amino group environments and metal binding properties of carbon-13 reductively methylated bovine alpha-lactalbumin

13C NMR spectroscopy has been used to study the amino group environments and metal binding properties of 13C reductively methylated bovine alpha-lactalbumin. Bovine alpha-lactalbumin is a Ca2+ metalloprotein containing 12 lysyl amino groups and a free amino terminus. All 13 amino groups can be 13C-dimethylated without altering Ca2+ binding or biological activity. pH titrations (chemical shift vs. pH) of this dimethylated protein reveal unique behavior for each of the 13 amino groups. The pKa values for the lysyl amino groups range from 9.1 to 10.8 while the pKa for the N-terminal amino group is 8.3. This relatively high pKa (by 1 pH unit) for the N-terminal supports its interaction in an ion pair as proposed by Warme et al. [Warme, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., & Scheraga, H. A. (1974) Biochemistry 13, 768-782]. Carbon-13 NMR studies further show that the removal of Ca2+ from the high-affinity binding site results in a conformational change, with the disruption of the N-terminal ion pair interaction (pKa decreased to 7.4). The study of Zn2+ binding to Ca2+-saturated protein suggests that Zn2+ binds initially at a low-affinity Ca2+ site while maintaining the N-terminal ion pair interaction. The further addition of Zn2+ leads to the disruption of this ion pair forming a presumed apoprotein-like conformation. Finally on the basis of the specific effects of added Mn2+ on the 13C NMR spectra of the methylated protein, a low-affinity divalent metal binding site is proposed about 7.5 A from the amino terminus.     

Calcium-induced cooperativity of manganese binding to concanavalin A.

Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.     

Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: equilibrium studies.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)