scholarly journals Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase

1985 ◽  
Vol 225 (3) ◽  
pp. 573-580 ◽  
Author(s):  
P N B Gibbs ◽  
M G Gore ◽  
P M Jordan
Keyword(s):  
Metal Ions ◽  
Binding Sites ◽  
The Other ◽  
Stopped Flow ◽  
Thiol Groups ◽  
Protein Fluorescence ◽  
Fluorescence Studies ◽  
Nitrobenzoic Acid ◽  
Rate Of Reaction ◽  
Molar Equivalent

The reaction of human 5-aminolaevulinate dehydratase with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.

scholarly journals Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

1992 ◽  
Vol 285 (2) ◽  
pp. 419-425 ◽  
Author(s):  
U Christensen ◽  
L Mølgaard
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Change ◽  
Conformational Changes ◽  
Binding Sites ◽  
High Specificity ◽  
Stopped Flow ◽  
Protein Fluorescence ◽  
Lysine Residues ◽  
Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

scholarly journals Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli

1994 ◽  
Vol 300 (2) ◽  
pp. 373-381 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Metal Binding ◽  
Binding Sites ◽  
Metal Ion ◽  
Protein Fluorescence ◽  
Metal Binding Sites ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Aminolaevulinic Acid Dehydratase

Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli. The alpha-site binds a Zn2+ ion that is essential for catalytic activity. This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+. The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site. The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values. The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6. Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes. Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site. Reaction with N-ethyl[14C]maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

scholarly journals Rapid and selective modification of phosphoserine residues catalysed by Ba2+ ions for their detection during peptide microsequencing

1991 ◽  
Vol 280 (1) ◽  
pp. 261-265 ◽  
Author(s):  
M F Byford
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Metal Ions ◽  
Ion Concentration ◽  
Thiol Groups ◽  
Disulphide Bonds ◽  
Rate Of Reaction ◽  
Alkaline Conditions ◽  
Beta Elimination ◽  
Alpha Beta

The beta-elimination of phosphoserine residues by dilute alkali is catalysed by the presence of group II metal ions. The use of 0.1 M-Ba (OH)2 catalysed the rate of beta-elimination of phosphoserine by more than two orders of magnitude compared with the use of NaOH at the same OH-ion concentration. Serine and threonine residues are unaffected by this treatment. Free thiol groups and disulphide bonds are labile to these conditions, but carboxymethylcysteine is stable. The rate of beta-elimination of O-glycosidically linked moieties is not catalysed under these conditions, and the rate of reaction is thus two orders of magnitude slower than for phosphoserine. This specific catalysis was readily exploited in the rapid and selective modification of phosphoserine residues under mildly alkaline conditions with the nucleophile methylamine via the alpha beta-desaturated dehydroalanine intermediate to yield the beta-methylaminoalanine residue. This modified residue could be easily detected on sequence analysis and in amino acid compositions.

scholarly journals Calcium and thiol reactivity of human plasma clotting factor XIII

1974 ◽  
Vol 141 (3) ◽  
pp. 675-682 ◽  
Author(s):  
Rodney D. Cooke ◽  
Timothy C. Pestell ◽  
J. John Holbrook
Keyword(s):  
Enzyme Inhibition ◽  
Factor Xiii ◽  
Protein Concentration ◽  
Factor Xiiia ◽  
Clotting Factor ◽  
Stopped Flow ◽  
Thiol Groups ◽  
Thiol Reactivity ◽  
Rate Of Reaction ◽  
Very High

1. The reaction of iodoacetate, 2-chloromercuri-4-nitrophenol and 5,5′-dithiobis-(2-nitrobenzoate) with thrombin-cleaved Factor XIII (i.e. Factor XIIIa) was accompanied by enzyme inhibition. 2. The reaction with iodoacetate and 5,5′-dithiobis-(2-nitrobenzoate) was absolutely dependent on Ca2+, and the rate of reaction increased with the Ca2+concentration up to very high, non-physiological concentrations. 3. 2-Chloromercuri-4-nitrophenol reacted with Factor XIIIa in the absence of Ca2+, but at a much slower rate. 4. Stopped-flow methods were used to quantify the reaction with 5,5′-dithiobis-(2-nitro-benzoate) because of the Ca2+-dependent dissociation of Factor XIIIa (a′2b2) and subsequent aggregation of the a′ chains into turbid precipitates. 5. The 3-carboxy-4-nitrothio-phenolate released was consistent with the reaction of 2 thiol groups/molecule of Factor XIIIa. The isolated b chains of Factor XIII did not react with either of the chromophoric reagents. This indicated that the a′ chains of Factor XIIIa were responsible for the thiol reactivity of the enzyme. 6. The Ca2+dependence of the enzyme inhibition by these thiol reagents was very dependent on protein concentration. This is discussed in relation to the Ca2+-induced dissociation of Factor XIIIa. 7. The acceptor substrate, casein, decreased the Ca2+concentration required for enzyme inhibition by both the mercurial and the aromatic disulphide compounds. Dansylcadaverine did not affect Ca2+dependence of inhibition.

scholarly journals Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315

1976 ◽  
Vol 155 (1) ◽  
pp. 37-53 ◽  
Author(s):  
R A Dwek ◽  
D Givol ◽  
R Jones ◽  
A C McLaughlin ◽  
S Wain-Hobson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Spin Label ◽  
Binding Sites ◽  
Binding Constants ◽  
Kinetic Scheme ◽  
Difference Spectrum ◽  
Protein Fluorescence ◽  
Myeloma Protein ◽  
Fluorescence Studies

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a ‘slow’ conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment.

THE MOLECULAR ORGANIZATION OF HUMAN ALPHA 2-MACROGLOBULIN. AN IMMUNO ELECTRON MICROSCOPIC STUDY WITH MONOCLONAL ANTIBODIES

1987 ◽  
Author(s):  
E Delain ◽  
M Barrav ◽  
J Tapon-Bretaudière ◽  
F Pochon ◽  
F Van Leuven
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Sites ◽  
Divalent Cations ◽  
The Other ◽  
Molecular Organization ◽  
Cellular Receptor ◽  
Electron Microscopic ◽  
Microscopic Method ◽  
Bait Region ◽  
Electron Microscopic Method

Electron microscopy is a very convenient method to localize the epitopes of monoclonal antibodies (mAbs) at the surface of macromolecules for studying their tree-dimensional organization.We applied this immuno-electron microscopic method to human ct2-macroglobulin (ct2M). 29 anti-α2M mAbs have been tested with the four different forms of a2M : native and chymotrypsin-transformed tetramers, and the corresponding dimers, obtained by dissociation with divalent cations. These mAbs can be classified in three types : those which are specific for 1) the H-like transformed molecules, 2) the native molecules, and 3) those which can react with both forms of α2M.1) Among the H-like α2M specific mAbs, several react with the 20 kD-domain which is recognized by the cellular receptor of transformed a2M. This domain is located at the carboxyterminal end of each monomer. One IgG binds to the end of two adjacent tips of the H-like form.The other mAbs of this type bind to the α2M tips at non-terminal positions. Intermolecular connections built polymers of alternating α2M and IgG molecules.2) Among the native a2M-specific mAbs some are able to inhibit the protease-induced transformation of the native α2M. The binding sites of these mAbs are demonstrated on the native half-molecules. One of these mAbs was also able to react with transformed dimers, in a region corresponding very likely to an inaccessible epitope in the tetrameric transformed α2M molecule.3) Among the mAbs of this type, only two were able to inhibit the protease-induced transformation of α2M. Obviously, their epitopes should be close to the bait region of α2M. The other mAbs reacting with both α2M forms did not inhibit the α2M transformation.All these mAbs can be distinguished by the structure of the immune complexes formed with all forms of α2M. The epitopes are more easily located on the dimers and on the H-like transformed α2M than on the native molecules.From these observations, we propose a new model of the tree-dimensional organization of the human α2M in its native and transformed configurations, and of its protease-induced transformation.

The influence of metal-ion binding on the structure and surface composition of Sonic Hedgehog: a combined classical and hybrid QM/MM MD study

2016 ◽  
Vol 18 (32) ◽  
pp. 22254-22265 ◽  
Author(s):  
Manuel Hitzenberger ◽  
Thomas S. Hofer
Keyword(s):  
Metal Ions ◽  
Sonic Hedgehog ◽  
Binding Sites ◽  
Metal Ion ◽  
Surface Composition ◽  
Quantum Mechanical ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Structural Impact

The interaction of metal ions with Shh binding-sites and their structural impact are assessed via classical and quantum mechanical simulations.

scholarly journals Effects of dl-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites

1985 ◽  
Vol 230 (1) ◽  
pp. 169-179 ◽  
Author(s):  
M R Edwards ◽  
M I Bird ◽  
E D Saggerson
Keyword(s):  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Heart Mitochondria ◽  
Nitrobenzoic Acid ◽  
Malonyl Coa ◽  
Tissue Differences ◽  
The Relationship

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5′-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.

Sign in / Sign up

Export Citation Format

Share Document