The structure of bovine liver rhodanese. II. The active site in the sulfur-substituted and the sulfur-free enzyme.

X-ray studies at 2.5 Å resolution show that the active site of bovine liver rhodanese is a depression between the two domains. In sulfur-substituted rhodanese the density of the essential Cys247 corresponds with that of a persulfide. Both sulfur atoms are interacting via hydrogen bonds with several peptide NH and side-chain OH groups. One side of the active site pocket contains mainly hydrophylic, the other side mainly hydrophobic residues. None of these hydrophylic or hydrophobic groups appears to interact strongly with the persulfide.Crystals of the sulfur-substituted enzyme were treated with cyanide, a sulfur acceptor. Subsequent difference Fourier studies show that the extra sulfur atom has been removed. Only minor conformational differences appear to exist between the two rhodanese species studied. These are a movement of the Sγ atom of Cys247 and some rearrangement of solvent molecules near the active site.The combination of these observations with the results of experiments performed by other investigators suggest a mechanism for sulfur transfer by rhodanese in which the thiol group of Cys247 is the essential nucleophile, whereas the positive charges on Arg186 and Lys249 act in various ways as “electrophilic assistants”. The transition state and the persulfide in the sulfur-substituted enzyme are stabilized by several hydrogen bonds.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.     

An interaction between selenate and a sulfate transporter in the lactating rat mammary gland.

The efficacy of selenate and other divalent anions to stimulate the efflux of radiolabeled sulfate from lactating rat mammary tissue slices has been examined. Both selenate and sulfate markedly increased the fractional release of sulfate via a system that is temperature-sensitive and sensitive to the anion-exchange inhibitor DIDS. The effect of selenate on sulfate efflux was saturable with an apparent affinity constant of approximately 0.27 mM. In addition, molybdate and thiosulfate were also found to increase sulfate efflux from the trans-aspect. It is concluded that sulfate and selenate share a pathway for transport in the lactating rat mammary gland.     

On the size and chemical nature of the polypeptide chain of bovine liver rhodanese.

Evidence from molecular weight studies and sequence analysis of bovine liver rhodanese indicates that the enzyme is a single polypeptide of molecular weight 35,200, and not a dimer of identical subunits half this size. The rhodanese molecule contains 317 amino acids including 5 methionines, 4 cysteines, and 5 tryptophans. As expected, six fragments were produced by cleavage with cyanogen bromide and these have been aligned in the enzyme with the aid of overlapping tryptic peptides isolated from a [¹⁴C] carboxymethylmethionyl rhodanese derivative. The cyanogen bromide fragments account for all of the amino acid residues of the parent rhodanese molecule. Methionine residues are located at positions 72, 112, 214, 217, and 235 in the polypeptide chain and the active site cysteine is at position 251, in the carboxyl-terminal segment of the molecule.     

In vitro study on the binding of anti-coagulant vitamin to bovine serum albumin and the influence of toxic ions and common ions on binding

The mechanism of binding of vitamin K(3) (VK(3)) with bovine serum albumin (BSA) was investigated by fluorescence, absorption and circular dichroism (CD) techniques under physiological conditions. The analysis of fluorescence data indicated the presence of static quenching mechanism in the binding. Various binding parameters have been evaluated. Thermodynamic parameters, the standard enthalpy change, DeltaH(0) and the standard entropy change, DeltaS(0) were observed to be -164.09 kJ mol(-1) and -465.08 J mol(-1)K, respectively. The quantitative analysis of CD spectra confirmed the change in secondary structure of the protein upon interaction with VK(3). The binding average distance, r between the donor (BSA) and acceptor (VK(3)) was determined based on the F?rster's theory and it was found to be 3.3 nm. The effects of toxic ions and common ions on VK(3)-BSA system were also investigated.     

Evidence in liver for a disulphide-linked scavenger receptor containing a binding site for acetylated low-density lipoprotein and maleylated bovine serum albumin.

Membranes from rat liver were analysed under reducing conditions. The components of the soluble membranes responsible for the binding of acetylated low density lipoprotein (acetyl-LDL) and maleylated bovine serum albumin (Mal-BSA) were chromatographed on a polyethyleneimine-cellulose column and subsequently separated by gel electrophoresis. For both ligands a major binding protein (Mr = 35,000) was revealed by ligand blotting. A minor protein (Mr greater than 67,000) exhibited little binding. The Scatchard plot of the 131I-Mal-BSA binding data of the 35 kDa protein was linear, with a Kd of 17.3 nM. High concentrations of acetyl-LDL competed for half of the 131I-Mal-BSA binding. Excessive Mal-BSA competed for all the visible acetyl-LDL binding. The findings indicate the existence, in the reduced hepatic membrane, of a 35 kDa protein that has two binding sites for 131I-Mal-BSA and one binding site for acetyl-LDL.     

Alkyl isocyanates as active site-specific reagents for serine proteases. Location of alkyl binding site in chymotrypsin by X-ray diffraction.

The structure of octylcarbamoyl-alpha-chymotrypsin to a resolution of 3.0 A is described. The n-octyl side chain of the active site directed irreversible inactivator octyl isocyanate is bound exclusively in the hydrophobic substrate binding pocket. The n-octyl isocyanate forms a planar urethane bond with the Ser-195 Ogamma and extends approximately 1 A deeper into the hydrophobic pocket than the indolyl group of indoleacryloyl-alpha-chymotrypsin (Henderson, R. (1970), J. Mol. Biol. 54, 341). All the structural changes are essentially identical with those observed in indoleacryloyl-alpha-chymotrypsin including the observation of a hydrogen bonded water molecule between the carbonyl oxygen of the octylcarbamoyl group and the imidazole group of His-57. The observed mode of n-octyl alkyl binding to chymotrypsin is consistent with the hypothesis proposed earlier (Brown, W. E. and Wold, F. (1973), Biochemistry 12, 828).     

NMR analysis of the binding of a rhodanese peptide to a minichaperone in solution.

A detailed structural analysis of interactions between denatured proteins and GroEL is essential for an understanding of its mechanism. Minichaperones constitute an excellent paradigm for obtaining high-resolution structural information about the binding site and conformation of substrates bound to GroEL, and are particularly suitable for NMR studies. Here, we used transferred nuclear Overhauser effects to study the interaction in solution between minichaperone GroEL(193-335) and a synthetic peptide (Rho), corresponding to the N-terminal alpha-helix (residues 11 to 23) of the mitochondrial rhodanese, a protein whose in vitro refolding is mediated by minichaperones. Using a 60 kDa maltose-binding protein (MBP)-GroEL(193-335) fusion protein to increase the sensitivity of the transferred NOEs, we observed characteristic sequential and mid-range transferred nuclear Overhauser effects. The peptide adopts an alpha-helical conformation upon binding to the minichaperone. Thus the binding site of GroEL is compatible with binding of alpha-helices as well as extended beta-strands. To locate the peptide-binding site on GroEL(193-335), we analysed changes in its chemical shifts on adding an excess of Rho peptide. All residues with significant chemical shift differences are localised in helices H8 and H9. Non-specific interactions were not observed. This indicates that the peptide Rho binds specifically to minichaperone GroEL(193-335). The binding region identified by NMR in solution agrees with crystallographic studies with small peptides and with fluorescence quenching studies with denatured proteins.     

On the competition between water,sodium ions,and spermine in binding to DNA: a molecular dynamics computer simulation study

The interaction of DNA with the polyamine spermine(4+) (Spm(4+)), sodium ions, and water molecules has been studied using molecular dynamics computer simulations in a system modeling a DNA crystal. The simulation model consisted of three B-DNA decamers in a periodic hexagonal cell, containing 1200 water molecules, 8 Spm(4+), 32 Na(+), and 4 Cl(-) ions. The present paper gives a more detailed account of a recently published report of this system and compares results on this mixed Spm(4+)/Na(+)-cation system with an molecular dynamics simulation carried out for the same DNA decamer under similar conditions with only sodium counterions (Korolev et al., J. Mol. Biol. 308:907). The presence of Spm(4+) makes significant influence on the DNA hydration and on the interaction of the sodium ions with DNA. Spermine pushes water molecules out of the minor groove, whereas Na(+) attracts and organizes water around DNA. The major binding site of the Spm(4+) amino groups and the Na(+) ions is the phosphate group of DNA. The flexible polyamine spermine displays a high presence in the minor groove but does not form long-lived and structurally defined complexes. Sodium ions compete with Spm(4+) for binding to the DNA bases in the minor groove. Sodium ions also have several strong binding sites in the major groove. The ability of water molecules, Spm(4+), and Na(+) to modulate the local structure of the DNA double helix is discussed.     

Fibre X-ray and conformational study of the binding of metal ions on DNA

Results obtained from X-ray diffraction as well as from conformational analysis of Ag-DNA fibres are presented. For small percentages of Ag+ bound and high humidity, the B-DNA form is maintained. As the percentage of Ag+ is increased, the helical parameters of the B-DNA are modified. These modifications are directly related to the percentage of G-C bases. The periodicity of the DNA fibres are perturbed as Ag+ is mainly bound to G-C pairs and, thus, only the equatorial diffracted intensities can be compared to values calculated from molecular models. It is shown, by this way, that the first binding site is located on N7 of G. A second site is situated between N3 and N1 of the G-C pair, at the place of a hydrogen bond. A molecular model of the Ag-DNA complex is proposed and shown to be in agreement with experimental data. Results obtained allow to get some information on the binding of other ions such as Cu2+ and Hg2+ which give very little modification of the fibre X-ray patterns.     

Spectroscopic investigations on the binding site of bovine hepatic fatty acid binding protein. Evidence for the existence of a single binding site for two fatty acid molecules

The hydrophobic region of the binding site of a bovine fatty acid binding protein (pI 7.0-FABP) has been characterized using fluorescence and circular dichroism (CD) spectroscopy. Blue-shifts of fluorescence emission maxima and increased lifetimes of naphthylamine dyes, anthroyloxy-fatty acids, pyrene nonanoic acid and trans-parinaric acid indicated a hydrophobic interaction with FABP. The fluorescence quenching of various anthroyloxy-fatty acids by iodide and acrylamide showed lower accessibility to the fluorophore linked to the carbon adjacent to the carbonyl group and towards the methyl end of the fatty acid. Binding stoichiometries were different for fatty acids and their bulky fluorescent analogues. trans-Parinaric acid when bound to FABP showed a complex induced CD-spectrum, which is explained by a close proximity of two ligands in the same binding site. Fluorescent derivatives of phosphatidylcholine with trans-parinaric acid and cholesteryl trans-parinarate did not bind to FABP. Thus, the binding site appears to be constructed for high affinity binding of long chain fatty acids.     

Response of rat liver glutaminase to pH. Mediation by phosphate and ammonium ions

The activity of rat liver glutaminase from sedimented fractions of freeze-thawed mitochondria is strongly affected by variation in pH over a physiologically relevant range at approximate physiological concentrations of activators. As pH increases from 7.1 to 7.7 at 0.7 mM ammonium and 10 mM phosphate, the S0.5 for glutamine decreases 3.5-fold, from 38 to 11 mM. This results in an 8-fold increase in reaction velocity at 10 mM glutamine. In addition, the M0.5 for phosphate activation decreases from 21 to 8.9 mM as pH increases from 7.1 to 7.7. This apparent effect of pH on the affinity of glutaminase for phosphate is similar to previous reports of the pH effect on activation by ammonium (Verhoeven, A. J., Van Iwaarden, J. F., Joseph, S. K., and Meijer, A. J. (1983) Eur. J. Biochem. 133, 241-244; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 159, 296-302). Glutaminase does not respond to variation in pH between 7.1 and 7.7 when phosphate and ammonium are saturating. The effects of the two modifiers are additive. Each is still effective, as is pH, when the other is saturating. Therefore, it appears that the effects of pH on the apparent affinity of the enzyme for ammonium and phosphate account for the enzyme's response to pH. These results may help explain previous reports of minimal effects of pH on glutaminase at saturating concentrations of related substances (McGivan, J. D., Lacey, J. H., and Joseph, K. (1980) Biochim. J. 192, 537-542; Horowitz, M. L., and Knox, W. E. (1968) Enzymol. Biol. Clin. 9, 241-255; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 759, 296-302). Glutaminase binds glutamine cooperatively with Hill coefficients ranging from 1.7 to 2.2, which suggests at least two and probably three or more interacting binding sites for glutamine. The strong response of liver glutaminase to pH and the fact that the reaction can supply metabolites for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Human liver cathepsin D. Purification, crystallization and preliminary X-ray diffraction analysis of a lysosomal enzyme.

The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.     

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.     

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

• 1.1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with K_i = 11 nM.
• 2.2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme.
• 3.3. The dissociation constants (K_d) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations.
• 4.4. The K_d values for NADPH, MTX and FMTX from the complementary binary complexes (MTX·DHFR, FMTX·DHFR and NADPH·DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme.
• 5.5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10⁻⁹ to 10⁻⁷ M.